Decoding asthma: Translating genetic variation in IL33 … · Decoding asthma: Translating genetic variation in IL33 and ... the gene encoding the transcription factor critical for

Decoding asthma: Translating genetic variation in IL33 andIL1RL1 into disease pathophysiology

N�eomi S. Grotenboer, MSc,a,b,c Maria E. Ketelaar, MSc,a,c Gerard H. Koppelman, MD, PhD,b,c* and

Martijn C. Nawijn, PhDa,c* Groningen, The Netherlands

Abbreviations used

ASW: African ancestry in Southwest United States

CEU: Utah residents with Northern and Western European

ancestry from the CEPH collection (referred to as

non-Hispanic white)

CHB: Han Chinese in Beijing, China

GWA: Genome-wide association

ILC2: Type 2 innate lymphoid cell

IL1RL1: IL-1 receptor–like 1

IL18R1: IL-18 receptor 1

LD: Linkage disequilibrium

MEX: Mexican ancestry in Los Angeles, California (referred

to as Mexican population)

MyD88: Myeloid differentiation primary response gene–88

NF-kB: Nuclear factor kB

RORA: Retinoic acid–related orphan receptor a

SNP: Single nucleotide polymorphism

TIR: Toll-like/IL-1 receptor

TLR: Toll-like receptor

YRI: Yoruban in Ibadan, Nigeria

Asthma is a complex disease that results from the interactionbetween genetic predisposition and environmental factors.Recently, genome-wide association studies have identified anumber of genes that significantly contribute to asthma. Two ofthese genes, IL33 and IL-1 receptor–like 1 (IL1RL1), act in onesignal transduction pathway. IL33 encodes a cytokine releasedon damage of cells, whereas IL1RL1 encodes part of the IL-33receptor complex. Recent progress made in functional studies inhuman subjects and mouse models of allergic airway diseaseindicate a central role of IL-33 signaling in driving TH2inflammation, which is central to eosinophilic allergic asthma.Here, IL-33 acts on cells of both the adaptive and innateimmune systems. Very recently, a novel population of IL-33–responsive innate immune cells, the type 2 innate lymphoid cells,was found to produce hallmark TH2 cytokines, such as IL-5 andIL-13. The relevance of these cells for asthma is underscored bythe identification of retinoic acid–related orphan receptor a(RORA), the gene encoding the transcription factor critical fortheir differentiation, as another asthma gene in genome-wideassociation studies. This review describes the mechanismsthrough which genetic variation at the IL33 and IL1RL1 locitranslates into increased susceptibility for asthma. We proposethat genetic variation associated with asthma at the IL33 andIL1RL1 loci can be dissected into independent signals withdistinct functional consequences for this pathway that iscentral to asthma pathogenesis. (J Allergy Clin Immunol2013;131:856-65.)

Key words: IL-33, IL-1RL1, ST2, genome-wide association study,nuocytes, innate helper cells, innate type 2 lymphoid cells,expression quantitative trait locus

From athe Laboratory of Allergology and Pulmonary Diseases, Department of Pathology

andMedical Biology, and bthe Department of Pediatric Pulmonology and Pediatric Al-

lergology, Beatrix Children’s Hospital, and cGRIAC Research Institute, University of

Groningen, University Medical Center Groningen.

*These authors contributed equally to this work.

Supported by a Netherlands Asthma Foundation grant (no. 3.2.09.081JU).

Disclosure of potential conflict of interest: G. H. Koppelman has been supported by one or

more grants from or has one or more grants pending with the Netherlands Asthma

Foundation, the EuropeanUnion, andStichtingAstmaBestrijding and has received one

or more payments for lecturing from or is on the speakers’ bureau for GlaxoSmithK-

line. M. C. Nawijn has received one or more grants from or has one or more grants

pending with the Netherlands Asthma Foundation, the European Union, and Stichting

Astma Bestrijding, and has received support for travel from the European Respiratory

Society. The rest of the authors declare that they have no relevant conflicts of interest.

Received for publication July 24, 2012; revised October 30, 2012; accepted for publica-

tion November 19, 2012.

Available online February 4, 2013.

Corresponding author: Martijn C. Nawijn, PhD, Laboratory of Allergology and Pulmo-

nary Diseases, Pathology andMedical Biology, IPC EA11, University Medical Center

Groningen, GRIAC Research Institute, University of Groningen, Hanzeplein 1, PO

Box 30.001, 9700 RB, Groningen, The Netherlands. E-mail: [email protected].

0091-6749/$36.00

� 2013 American Academy of Allergy, Asthma & Immunology

http://dx.doi.org/10.1016/j.jaci.2012.11.028

856

Asthma is a complex, chronic inflammatory disease of the
airways currently affecting more than 300 million personsworldwide, with approximately 250,000 annual deaths as aresult.1 It is estimated that by 2025, the number of asthmaticpatients will increase by more than 100 million.2
Asthma is characterized by respiratory symptoms, variableairway obstruction, and airway hyperresponsiveness. The clinicalexpression of asthma is dependent on the interaction betweengenetic predisposition and environmental factors. The number ofidentified asthma susceptibility genes has increased rapidly overthe last 5 years, especially with the application of the genome-wide association (GWA) study approach. In a GWA study300,000 to more than a million DNA polymorphisms coveringthe genome are investigated for association with asthma in largesamples of cases and control subjects.Genetic variation in the IL33 and IL-1 receptor–like 1 (IL1RL1)

genes has reproducibly been found to be associated with asthmain GWA studies, identifying IL-33–induced signaling throughIL-1RL1 as one of the central pathways in asthmatic patients. Al-though numerous functional studies have revealed a central rolefor IL-33–induced signaling in TH2-driven inflammation, whichplays a crucial role in allergic asthma, few studies have directlyassessed the functional consequences of genetic variation in theIL33 and IL1RL1 genes for the activity of this pathway. Such stud-ies are currently limited by the large number of asthma-associatedsingle nucleotide polymorphisms (SNPs) in these genes and thecomplex genetic structure of the IL1RL1 locus.

Therefore this review aims to offer an interpretation of asthma-associated polymorphisms in IL33 and IL1RL1 as a limitednumber of discrete genetic signals with distinct functional conse-quences and to discuss these in the context of a newly identified

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http://dx.doi.org/10.1016/j.jaci.2012.11.028

J ALLERGY CLIN IMMUNOL

VOLUME 131, NUMBER 3

GROTENBOER ET AL 857

population of innate IL-33–responsive, IL-1RL11 immune cells,the type-2 innate lymphoid cells (ILC2s), in the pathophysiologyof asthma.

GENETIC ASSOCIATION OF THE IL-33/IL-1RL1

PATHWAY WITH ASTHMA SUSCEPTIBILITYIL-33 is an IL-1 family member and the ligand of the IL-1RL1

(ST2) receptor. The IL33 genewas initially found to be associatedwith Cedar pollinosis in a Japanese population.3 One year later, aGWA study in an Icelandic population identified SNPs flankingIL33 to be suggestively associated with blood eosinophils.4

This finding was followed up by a large case-control study ofasthmatic patients and control subjects, indicating significantassociation of IL33 SNPs with asthma.4 Subsequent GWA studymeta-analyses by the European GABRIEL consortium,5 whichwas recently combined with the Analysis in Population-basedCohorts for Asthma Traits consortium meta-analysis,6 and theNorth American EVE consortium7 identified IL33 as one of thetop hits for asthma.Eight IL33 SNPs have been reported to be associated with

asthma phenotypes (Table I and see the Methods section in thisarticle’s Online Repository at www.jacionlone.org).4-15 Severalof these SNPs are in close proximity to each other and areobserved more frequently together than would be expected bychance based on the allele frequencies in the population. Thisphenomenon is termed linkage disequilibrium (LD) and indicatesthat the SNPs are correlated in populations. In case the SNPs arein full LD, they are always inherited together and therefore repre-sent a single genetic signal. A series of such highly correlatedSNPs form an LD block, and their alleles form a haplotype.Although a single SNP within this LD block might be responsiblefor the functional alteration that contributes to disease, all SNPswithin the LD block will be associated with asthma in a geneticassociation analysis. When the LD structure of IL33 in the Utahresidents with Northern and Western European ancestry fromthe CEPH collection (CEU; referred to as non-Hispanic white)ethnic background, in which most association analyses havebeen performed, is taken into account (Fig 1, A), it becomesapparent that 4 of the asthma-associated SNPs are located in 2LD blocks, whereas the remaining 4 asthma-associated SNPsare not in strong LD with other polymorphisms (Table II [andsee the Methods section in this article’s Online Repository] andFig 1, A).4-21 Interestingly, in the Yoruban in Ibadan, Nigeria(YRI) population and, to some extent in the African ancestry inSouthwest United States (ASW) and Han Chinese in Beijing(CHB) ethnic backgrounds, the LD patterns of IL33 are different(see Fig E1 in this article’s Online Repository at www.jacionline.org), in that the SNPs clustering into LD blocks within the CEUpopulation are independent SNPs in the studied African andAsian populations, indicating that association analyses in patientsof these ethnic backgrounds might reveal the causal SNP retainedwithin the 2 LD blocks.IL1RL1 was first described as a candidate gene for atopic

dermatitis.22 Our group was the first to report IL1RL1 as anasthma susceptibility locus by taking a candidate gene ap-proach.10 In total, 7 candidate gene studies4,9-14 and 4 GWAstudies5,6,7,16 have reported an association of IL1RL1 SNPswith asthma.Thus far 15 different IL1RL1 SNPs have been reported to be

associated with asthma (Table I). These SNPs lie scattered

throughout the IL1RL1 gene (Fig 1, B). Analysis of the LD struc-ture of IL1RL1 and its surrounding genomic region in patients ofthe CEU ethnic background reveals a complex LD pattern inwhich 5 LD blocks containing asthma SNPs and 1 independentasthma SNP can be distinguished, each of which might sepa-rately contribute to asthma susceptibility (Table II). Importantly,several SNPs in IL1RL1 are in LD with SNPs in the genes encod-ing IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory pro-tein, 2 genes juxtaposed to IL1RL1 on chromosome 2q.10,24 Thusgenetic associations in the CEU population cannot conclusivelydetermine which of these genes is implicated in asthma. More-over, analysis of the LD pattern of IL1RL1 in different ethnicbackgrounds reveals that the LD structure of the IL1RL1/IL18R1 locus remains highly complex in all these populations(see Fig E2 in this article’s Online Repository at www.jacionline.org). The IL1RL1 SNP rs1041973 represents an inde-pendent SNP in populations of Africans, Asian, andMexican eth-nicity, whereas it is part of LD block 2 in non-Hispanic whitesubjects (Fig 1, B, and see Fig E2). In the Mexican ancestry inLos Angeles, California, population (MEX; Mexican population)2 SNPs, rs1041973 and rs13431828 (r2 5 0.66 in MEX; both inLD block 2 in CEU) were in fact both found to be significantlyassociated with asthma,11 indicating that there might be 2 inde-pendent asthma signals present within the non-Hispanic whiteLD block 2. Therefore association analyses in other ethnic back-grounds might aid the identification of the causal asthma-associated SNPs, but experimental validation will remain keyto proving the individual contribution of the identified causal var-iants. Potential experimental approaches include in vitro studieson the functional effects of asthma-associated haplotypes in pri-mary cells or in vivo studies using mouse models that have beenspecifically engineered to carry the risk or protective haplotypeof either IL1RL1 or IL18R1.

FUNCTIONAL GENETICS OF IL33 AND IL1RL1The IL33 gene, which is located on chromosome 9, spans

approximately 42.2 kb in length, harboring 8 exons. All IL33asthma-associated SNPs are located 59 of the gene or in the firstintron. Therefore it is tempting to speculate that these SNPs affectIL33 transcription and that asthma susceptibility alleles are asso-ciated with increased IL-33 production. At present, however, nopublished data support this function, warranting further explora-tion of the functional consequences of these SNPs.The IL1RL1 gene, which is located at chromosome 2q12, spans

approximately 40.5 kb in length, harboring 13 exons and a distaland a proximal promoter. The IL1RL1 gene encodes for proteinswith an extracellular region carrying 3 immunoglobulin-likedomains, a transmembrane domain, and an intracellular regionharboring a Toll-like/IL-1 receptor (TIR) domain. Three tran-scripts are expressed through alternative splicing: a short isoformencoding the soluble protein IL1-RL1-a (also called sST2); a longisoform encoding the full transmembrane receptor IL1-RL1-b(ST2L); and a less well-known variant that encodes a truncatedprotein with 2 immunoglobulin-like domains and a hydrophobictail called IL1-RL1-c (ST2V). Functionally, IL1-RL1-b acts totransduce the IL-33 signal to the intracellular compartment,whereas the soluble IL1RL1-a functions as a decoy receptor, cap-turing IL-33 and inhibiting its function.25,26

IL1RL1 asthma-associated SNPs can translate into functionalalterations of the IL-33/IL-1RL1 pathway through several

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http://www.jacionline.org




TABLE I. Asthma-associated polymorphisms located within the IL33 and IL1RL1 gene regions

SNP

Chromosome

position

Reported

allele SNP class

OR,* RR,yP value Study approach Population Reference

IL33

rs1342326 6.190.076 Cz

T (with

rs928413G)

59 1.20 (1.13-1.28),*

P 5 9.20E-10n

P 5 1.8E-02p

Meta-analysis (GWAS)

Candidate gene study,

2-locus haplotype

TDT

White (Eur, North-Am)

White (NE-Italy)

5

8

rs2381416 6.193.455 C 59 P 5 1.3E-06r Meta-analysis (GWAS) Mixed: North-Am

(Eur, Am, Afr-Am,

Afr-Car, Lat)

7

rs3939286 6.210.099 Az

A

59 1.12 (1.07-1.17),*

P 5 5.3E-06n

P 5 1.8E-03r

Candidate gene study


Mixed (Eur, E-Asia)

White (non-Hispanic)

4

15

rs928413 6.213.387 G (with

rs1342326T)

59 P 5 1.8E-02p Candidate gene study,

2-locus haplotype

TDT

White (NE-Italy) 8

rs2066362 6.219.176 Tz Intron 1.21 (1.13-1.29),*

P 5 1.39E-08nMeta-analysis (GWAS) White (Eur, North-Am) 5

rs16924159 6.229.417 Ay Intron 0.82 (0.68-0.99),*

P 5 3.7E-02tCandidate gene study White (non-Hispanic) 9

rs12551256 6.231.239 Gy Intron 0.82 (0.69-0.98),*

P 5 3.E-02tCandidate gene study White (non-Hispanic) 9

rs7025417 6.240.084 Cz Intron 1.23 (1.01-1.51),*

P 5 4.5E-02tCandidate gene study White (non-Hispanic) 9

IL1RL1

rs1420089 102.938.389 T Intron P 5 3.3E-02p� Candidate gene study White (Eur, North-Am) 12

rs3771180 102.953.617 G Intron P 5 1.93E-08n,r Meta-analysis (GWAS) Mixed: North-Am

(Eur, Am, Afr-Am,

Afr-Car, Lat)

7

rs13431828 102.954.653 Ty

Ty

C

59 UTR 0.45 (0.29-0.70),�P 5 2.0E-04o

0.63 (0.47-0.83),*

P 5 8.49E-04t

P 5 6.2E-03r and

P 52.7E-02r




Mexicans



11

9

15

rs13408661 102.955.082 Gz Intron 1.23 (1.15-1.31),*

P 5 1.1E-09n,vMeta-analysis (GWAS) White (Eur) 6

rs1041973 102.955.468 Ay

Ay

Ay

Coding,

nonsynonymous

0.58 (0.43-0.78),�P 5 3.5E-04o

0.80 (0.65-0.99),*

P 5 4.3E-02t

0.70 (0.54-0.91),*

P 5 8E-03q




Mexicans


White (Dutch)

11

9

13

rs10173081 102.957.348 Cz Intron 1.2 (1.11-1.29),*

P 5 1.42E-08nMeta-analysis (GWAS) Mixed: North-Am

(Eur, Am, Afr-Am,

Afr-Car, Lat)

7

rs1420101 102.957.716 Az

A

Coding,

synonymous

1.16 (1.11-1.21),*

P 5 5.5E-12n

P 5 3.9E-02p



Mixed (Eur, E-Asia)

White (Dutch)

4

10

rs1946131 102.961.929 T Intron P 5 1.5E-02p� Candidate gene study White (Eur, North-Am) 12

rs1921622 102.966.067 A Intron P 5 3.8E-02p Candidate gene study White (Dutch) 10

rs10197862 102.966.549 Gy Intron 0.75 (2),*

P 5 1.22E-02q

and 0.58 (2),*

P 5 1.5E-05s

Candidate gene study White (Aus-Eur) 14

rs1861246 102.966.783 G Intron P 5 5E-02p Candidate gene study White (Dutch) 10

rs1861245 102.966.906 G Intron P 5 3.2E-02p� Candidate gene study White (Eur, North-Am) 12

rs10204137 102.968.212 Gy

Gy

Coding,

nonsynonymous

0.87 (0.83-0.91),*

P 5 5.10E-09n

0.80 (0.67-0.97),*

P 5 2.1E-02t





5

9

(Continued)


MARCH 2013

858 GROTENBOER ET AL

TABLE I. (Continued)

SNP

Chromosome

position

Reported

allele SNP class

OR,* RR,yP value Study approach Population Reference

rs10192157 102.968.356 Ty

Ty

Coding,

nonsynonymous

0.87 (0.83-0.91),*

P 5 9.32E-09n

0.80 (0.67-0.97),*

P 5 2.1E-02t





5

9

rs10206753 102.968.362 Cy

Cy

Coding,

nonsynonymous

0.87 (0.83-0.91),*

P 5 6.84E-09n

0.80 (0.67-0.97),*

P 5 2.1E-02t





5

9

SNPs in the IL33 and IL1RL1 genes are reported to have a significant association with asthma, with ‘‘Y’’ representing the protective allele and ‘‘Z’’ representing the risk allele. The

significance level as a P value after nBonferroni correction or as a ofalse discovery rate q value level, puncorrected P value, or qcorrected P value; rin the replication cohort or sthe

follow-up study; as a tnominal P value; or vin the meta-analysis and replication combined. The Methods section in this article’s Online Repository describes Table I’s construction

method.

Afr-Am, African American; Afr-Car, African Caribbean; Am, American; Aus-Eur, Australian-European; E-Asia, East Asian; Eur, European; Lat, Latino; NE-Italy, Northeastern

Italy; North-Am, North American; TDT, transmission disequilibrium test; UTR, untranslated region.

*Odds ratio (OR) with 95% CI or � relative risk (RR) with 95% CI.

�Unlike the initial observed significance in the SLSJ study, the signal did not replicate in any of the other replication study cohorts (SAGE, CAPPS, or BHS).




mechanisms, such as changes in the level of expression or alteredprotein function through amino acid substitutions (Table II). Thedistal promoter region, for instance, carries several SNPs experi-mentally validated to affect IL1RL1 gene transcriptional activity.This was evidenced by studies using a Luciferase reporter in a hu-man mast cell line, showing that the haplotype consisting of theSNPs rs11685480 (G), rs6543115 (C), and rs6543116 (A) re-sulted in higher IL1RL1 transcriptional activity compared withthe AGG haplotype.22 Although the GCA haplotype was foundto be associated with atopic dermatitis22 and not with asthma,these studies do reveal the presence of functional SNPs withinthe promoter region of IL1RL1, making it worthwhile to investi-gate asthma-associated SNPs located within this region for theireffect on gene transcription as well. Further support for the tran-scriptional activity of these SNPs was provided in a Dutch birthcohort, in which the same alleles were associated with solubleIL-1RL1-a levels in serum.13

Within the complex LD pattern of the IL1RL1 gene, we identify5 LD blocks carrying asthma-associated SNPs (Fig 1, B). Each ofthese LD blocks constitutes a discrete genetic signal. Three ofthese IL1RL1 LD blocks (blocks 2, 3, and 5) will be discussedin detail based on their genome-wide significant associationwith asthma and their potential functional effects.The second LD block of IL1RL1 harbors multiple asthma-

associated polymorphisms, one of which has potential functionalimplications. SNP rs1041973 is located in exon 3, which encodesthe first immunoglobulin-like domain of the extracellular part ofthe IL-1RL1 protein. Rs1041973 is a nonsynonymous SNP that re-sults in an amino acid substitution from a neutral alanine residueinto a negatively charged glutamic acid residue. Lingel et al26 ele-gantly showed that amino acid substitutions affecting charged res-idues at the IL-33/IL-1RL1 binding interface strongly influencethe affinity of the IL-33/IL-1RL1 interaction, although the specificeffect of the amino acid substitution encoded by the rs1041973SNP was not tested. Nevertheless, it is tempting to speculate thatrs1041973 affects IL-33 binding to the IL-1RL1 protein.The third LD block contains the synonymous asthma SNP

rs1420101 in exon 5E, which is only retained in the transcriptencoding the truncated third isoform IL-1RL1-c. This mightindicate an effect of this SNP on this specific transcript. Surpris-ingly, an association of this SNP with IL18R1 mRNA levels hasbeen identified in human peripheral blood CD41 T lymphocytes,

identifying rs1420101 as an IL18R1 expression quantitative traitlocus.19 Thus this IL1RL1 SNP or an IL18R1 SNP in strong LDcould contribute to asthma development by altering IL18R1 tran-scription levels.19

The fifth LD block contains a large number of SNPs in full LDand is positioned at the 39 end of the gene around exon 11 andextends into the IL18R1 gene also harboring IL18R1 SNPs. FiveSNPs within this LD block have been found to be associatedwith asthma (Table II), 4 of which (rs10204137, rs10192157,rs10206753, and rs3771166) have been identified as expressionquantitative trait loci significantly associated with both IL1RL1mRNA levels in lymphoblasts19 and IL18R1 mRNA levels infat tissue.18,20,21,27 Therefore it can be hypothesized that theseSNPs contribute to asthma development by altering the transcrip-tion levels of either the IL1RL1 gene, the IL18R1 gene, or both.Further experimental evaluation is required to determine whichSNPs contained within this LD block represent causal variants af-fecting IL1RL1 or IL18R1 mRNA levels. Intriguingly, this LDblock also contains 5 nonsynonymous coding SNPs, 2 of whichaffect the same codon and thereby the same amino acid residue.The net result of these 5 nonsynonymous SNPs is a change of 4amino acids in the intracellular part of IL-1RL1 between therisk and the protective haplotype. These nonsynonymous SNPsare located within exon 11, which, together with exon 10, encodesfor the TIR domain of the intracellular part of the IL-1RL1 pro-tein. The TIR domain plays a crucial role in signal transductionbecause binding of IL-33 induces heterodimerization of IL-1RL1 with the coreceptor IL-1RAcP and subsequent interactionof the intracellular TIR domains of these 2 receptor subunitswith signaling adaptor proteins as myeloid differentiationprimary response gene–88 (MyD88) or Mal. This triggers the sig-naling cascade that eventually results in the activation of down-stream mitogen-activated protein kinases and transcriptionfactors, such as nuclear factor kB (NF-kB) and activator protein1 (Fig 2). Activation of this signaling pathway might modulatecytokine production and gene expression but also cell survival,differentiation, and motility, depending on the cell typeinvolved.25

The TIR/TIR domain interaction of IL-1RL1 and the adaptorproteins Mal and MyD88 has been modeled in considerabledetail.28 Interestingly, the T433 residue in IL-1RL1-b was identi-fied as an interfacing residue of the IL-1RL1/Mal complex.28

rs19

2999

6

37

69

71

9

71

71

71

26

18

1

0

0

0

0

23

3

19

2

1

2

0

3

12

13

13

14

12

13

4

13

13

1

1

37

68

71

9

71

71

71

25

19

1

1

0

0

0

23

2

19

3

1

2

0

4

12

13

13

14

12

13

4

13

13

0

1

37

67

71

10

71

71

71

26

18

1

0

0

0

0

23

3

19

2

2

3

0

3

10

13

13

13

11

13

3

13

13

0

1

50

52

2

52

52

52

57

5

1

6

10

10

10

11

4

6

13

14

14

7

14

17

7

7

7

8

7

9

7

7

6

3

95

3

95

95

95

37

12

0

4

0

0

0

22

1

17

0

0

0

2

0

25

7

7

8

9

7

7

7

7

0

1

3

39

15

0

5

0

0

0

20

1

20

0

0

0

2

0

26

9

9

9

10

9

6

9

9

0

0

3

3

3

2

3

0

1

2

2

2

2

1

1

9

9

13

2

14

6

3

3

3

0

3

5

3

3

1

27

39

15

0

5

0

0

0

20

1

20

0

0

0

2

0

26

9

9

9

10

9

6

9

9

0

0

39

15

0

5

0

0

0

20

1

20

0

0

0

2

0

26

9

9

9

10

9

6

9

9

0

0

39

15

0

5

0

0

0

20

1

20

0

0

0

2

0

26

9

9

9

10

9

6

9

9

0

0

12

3

6

2

2

2

11

4

10

6

6

6

7

6

8

4

4

4

4

4

3

4

4

6

0

3

11

17

16

17

74

1

58

17

18

18

12

18

26

18

18

18

16

18

5

18

18

10

5

1

16

19

17

0

1

4

1

4

1

1

1

2

2

2

2

2

2

0

2

2

1

19

64

59

64

8

38

17

48

46

46

88

46

25

6

6

6

6

6

7

6

6

75

2

13

36

26

59

62

58

72

56

17

2

2

3

3

2

5

2

2

64

1

13

32

27

57

60

56

68

54

16

1

1

2

2

1

4

1

1

59

1

13

36

26

59

61

56

72

56

16

2

2

2

3

2

4

2

2

63

1

1

43

17

18

17

9

18

29

24

24

28

26

24

13

24

24

8

4

6

27

27

26

52

26

12

7

7

7

7

7

10

7

7

63

2

35

39

30

19

30

54

41

41

41

44

41

14

41

41

16

8

95

55

95

4

7

7

7

8

7

0

7

7

47

24

95

52

95

6

9

9

9

11

9

0

9

9

46

24

52

3

8

8

8

6

8

0

8

8

47

23

52

28

7

7

7

8

7

9

7

7

88

3

3

8

8

8

5

8

0

8

8

45

23

35

35

36

38

35

47

35

35

24

11

95

71

10

0

95

71

10

0

95

73

10

0

95

68

95

95

11

0

71

10

0

71

71

14

25

10

0

10

0

2

rs47

4216

6

rs14

1242

6

rs

13

42

32

6

rs

23

81

41

6

rs18

8890

9

rs13

2840

60

rs99

2969

rs

39

39

28

6

rs

92

84

13

rs

20

66

36

2

rs16

9241

44

rs11

7944

19

rs22

1046

4

rs10

4358

16

rs10

9754

97

rs47

4084

0

rs

16

92

41

59

rs16

9241

61

rs

12

55

12

56

rs70

3325

8

rs70

3472

0

rs13

75

rs

70

25

41

7

rs47

4217

0

rs70

1957

5

rs10

9755

16

rs13

3038

3

rs19

2999

2

rs11

1357

3

rs10

9755

19

rs14

1242

1

rs70

4792

1

rs10

4827

4

rs10

8153

97

rs20

2699

1

1 2 3 4 5 6 7 8

IL33-variant 3 mRNA

IL33-variant 2 mRNA

IL33-variant 1 mRNA

Block 1 Block 2 Block 2 Block 2

A

FIG 1. LD of the polymorphisms located within the IL33 region (chromosome 9) and the IL1RL1/IL18R1region (chromosome 2). A, LD plot of the IL33 region in the CEU population (see the Methods section in

this article’s Online Repository for more details). B, LD plot of the IL1RL1/IL18R1 region in the CEU popula-

tion (see the Methods section in this article’s Online Repository for more details). Asthma-associated poly-

morphisms are depicted in boldface, and expression quantitative trait loci are underlined.


MARCH 2013


Because the nonsynonymous coding SNP rs4988956 (LD block 5,Table II) results in a change of this polar threonine residue into anonpolar alanine residue,29 it can be hypothesized that this SNPhas a direct effect on the affinity of the interaction between theTIR domains of IL-1RL1-b and Mal. This might directly affectIL-33–induced signaling through IL-1RL1. These data warrantfurther evaluation of the effect of these 4 amino acid substitutionsfor IL-33–induced activation of downstream signaling. We pro-pose that amino acid substitutions contained within the TIR do-main of IL-1RL1 affect coupling of IL-1RL1 to the coreceptorIL-1RAcP and adaptor proteins, such as Mal, and thus affectdownstream signaling.In addition to direct effects on IL-33–induced signaling, the

TIR domain–dependent interaction of IL-1RL1-b with the adap-tor proteins MyD88 and Mal also plays a role in the inhibition ofToll-like receptor (TLR) signaling by IL-1RL1 (Fig 2).30,31 IL-1RL1/Mal orMyD88 complex formationmay prevent the interac-tion betweenMyD88,Mal, or bothwith activated TLRs.28,30 Thusthe presence of multiple amino acid substitutions within the TIRdomain of IL-1RL1-b could result in altered inhibition of TLR ac-tivation. This possibility is especially intriguing given the genetic

interaction we have previously reported for IgE sensitizationbetween polymorphisms in IL1RL1 and TLR4.32

In summary, the IL1RL1 gene carries multiple independent ge-netic signals with possible functional consequences that arehighly divergent, including effects on expression of IL1RL1 andits neighboring gene IL18R1, as well as effects on IL-33/IL-1RL1 binding affinity, TIR domain interactions, and downstreamsignaling.

FROM GENETICS TO BIOLOGY: IL-33/IL-1RL1

SIGNALING IN THE PATHOPHYSIOLOGY OF

ASTHMAGenetic studies strongly implicate the IL-33/IL-1RL1 pathway

in asthmatic patients. This is further corroborated by mechanisticstudies in experimental animal models of asthma and clinicalstudies, which will be reviewed below. Very recently, it has beenshown that the IL-33/IL-1RL1 pathway can act through theactivation of a novel subset of innate immune cells called ILC2sor nuocytes that produce cytokines such as IL-5 and IL-13 in thelungs.33-35

rs11

6854

24

rs12

7121

35

rs11

1239

18

rs10

1826

39

rs11

6904

43

rs

14

20

08

9

rs14

2008

8

rs11

1239

20

rs67

0684

4

rs19

9746

7

rs12

7121

41

rs

37

711

80

rs

13

43

18

28

rs13

4085

69

rs

13

40

86

61

rs

10

41

97

3

rs

10

17

30

81

rs37

3212

9

rs

14

20

10

1

rs37

7117

5

rs12

7121

42

rs

19

46

13

1

rs65

4311

9

rs13

0289

93

rs13

0174

55

rs17

6963

76

rs12

9995

42

rs

19

21

62

2

rs

10

19

78

62

rs

18

61

24

5

rs49

8895

5

rs

10

20

41

37

rs

10

19

21

57

rs

10

20

67

53

rs48

5156

7

rs12

1058

08

rs37

5527

6

rs13

6234

8

Block 1 Block 2 Block 5

42 0153 5E 6 7 8 1191b

1a1a

Distal

promoter

Proximal

promoter

Block 3 Block 4 Block 2

31

35

37

34

34

3

33

33

31

31

32

6

8

8

8

6

8

34

30

8

22

1

22

1

22

3

1

10

6

2

2

2

2

2

3

3

2

2

2

95

10

96

13

16

16

16

21

16

54

59

16

42

0

42

0

42

10

0

16

13

5

5

5

5

5

10

10

5

5

5

95

95

95

10

95

95

95

95

91

14

18

18

18

23

18

48

54

18

37

0

37

0

37

10

0

13

14

3

3

3

3

3

10

10

3

3

3

10

95

9

13

13

13

16

13

60

65

13

45

1

45

0

45

9

0

21

9

4

6

6

6

6

9

9

6

6

6

10

95

11

15

15

15

18

15

57

62

15

42

1

42

0

42

9

0

19

11

4

6

6

6

6

9

9

6

6

6

11

95

10

14

14

14

17

14

59

63

14

44

1

44

0

44

9

0

20

10

5

7

7

7

7

9

10

7

7

7

10

8

10

10

10

1

1

1

1

2

1

5

6

1

8

1

8

1

8

1

17

1

19

19

19

19

19

19

19

19

95

12

15

15

15

20

15

54

61

15

43

0

42

0

42

8

0

15

12

4

6

6

6

6

8

9

6

6

6

95

13

16

16

16

21

16

52

59

16

41

0

40

0

40

7

0

13

13

3

4

4

4

4

7

7

4

4

4

96

13

16

16

16

21

16

53

59

16

42

0

42

0

42

9

0

16

13

5

6

6

6

6

9

9

6

6

6

96

13

16

16

16

21

16

53

59

16

42

0

42

0

42

9

0

16

13

5

6

6

6

6

9

9

6

6

6

13

17

17

17

21

17

56

62

17

44

0

44

0

44

10

0

14

13

6

6

6

6

6

10

10

6

6

6

90

90

90

74

90

5

5

90

7

1

7

1

7

1

1

15

82

17

17

17

17

17

1

1

17

17

17

82

4

5

6

0

6

0

6

1

0

14

90

15

15

15

15

15

1

1

15

15

15

82

4

5

6

0

6

0

6

1

0

14

90

16

15

15

15

15

1

1

15

15

15

82

4

5

6

0

6

0

6

1

0

14

90

16

15

15

15

15

1

1

15

15

15

82

6

1

82

2

0

2

0

2

2

0

7

74

15

15

15

15

15

2

2

15

15

15

4

5

6

0

6

0

6

1

0

14

90

16

15

15

15

15

1

1

15

15

15

91

4

72

2

67

2

67

5

2

33

5

25

30

30

30

30

5

5

30

30

30

5

80

0

80

0

80

6

0

37

5

33

33

33

33

33

6

6

33

33

33

6

0

6

0

6

1

0

14

90

15

15

15

15

15

1

1

15

15

15

13

13

8

13

46

7

41

41

41

41

41

8

8

41

41

41

13

13

1

6

1

5

5

5

5

5

1

1

5

5

5

14

7

14

46

7

37

41

41

41

41

7

7

41

41

41

14

1

6

1

5

5

5

5

5

1

1

5

5

5

7

14

46

7

37

41

41

41

41

7

7

41

41

41

1

17

1

19

19

19

19

19

19

19

19

6

1

5

5

5

5

5

1

1

5

5

5

15

3

5

5

5

5

17

18

5

5

5

17

17

17

17

17

1

1

17

17

17

19

20

19

19

19

19

19

19

19

19 19

19

19

19

19

19

rs11

6936

97

rs

37

711

66

Block 1 Block 1

IL18R1

42 3 5 6 7 81b

IL1RL1-a mRNA

42 0153 6 1187 91b

IL1RL1-b mRNA

42 3 5 5E 6 7 81b

IL1RL1-c mRNA

1a

1a

B

FIG 1. (Continued)




IL-33 has a dual effect as a transcription factor and a cytokine.First, IL-33 is capable of acting as a transcription factor, therebyaltering gene expression in a cell-autonomous, IL-1RL1-independent fashion. IL-33 can translocate to the nucleus andbind directly to the chromatin,36,37 as well as to the NF-kB pro-teins p50 and p65.38 In doing so, IL-33 is capable of regulatingthe expression of proinflammatory genes, such as IL6, IL8, andthe p65 subunit of NF-kB,39,40 and thusmight be a direct regulatorof the inflammatory response.Second, IL-33 signals through binding to the heterodimeric

receptor complex consisting of IL-1RL1 and the accessoryreceptor subunit IL-1RAcP.26,41 Binding of IL-33 to the IL-1RL1/IL-1RAcP heterodimeric receptor complex will recruit sig-naling adaptor proteins, such as Mal or MyD88. Mal and MyD88are signaling proteins that express a TIR domain42 through whichthey bind to the intracellular TIR domain of IL-1RL1-b and IL-1RAcP and subsequently trigger a signaling cascade culminatingin activation of mitogen-activated protein kinases and transcrip-tion factors, such as NF-kB and activator protein 1 (Fig 2). Severalrecent reviews offer detailed overview of IL-33–induced signaltransduction by IL-1RL1.25,43 IL-33 is constitutively expressedin lymphoid organs, epithelial barrier tissues, and the brain,

whereas its expression is induced in inflamed tissues and injuredepithelia, such as the lung alveoli, during papain-induced allergicairway inflammation.44 Because IL-33 is released on injury ordamage, it has been coined to act as an ‘‘alarmin,’’ translatingdamage into an activation of the inflammatory response.A large number of cell types relevant to asthma pathogenesis

have been shown to express IL-1RL1 and to be responsive toIL-33,45 including TH2 cells,46 mast cells,47,48 invariant naturalkiller T cells,49 eosinophilic and basophilic granulocytes,50-52

and epithelial cells.53 By activating these cells, IL-33 has beenshown to mediate a wide range of responses, including TH17-mediated airway inflammation54 and neutrophil influx,55 as wellas antiviral CD81 T-cell responses56 and lung tissue homeostasison influenza virus infection.57 However, the best described activ-ity of IL-33 is the activation of innate and adaptive immune re-sponses characterized by the production of IL-4, IL-5, and IL-13.In the first studies on its biological activities, systemic admin-

istration of IL-33 to mice induced production of IL-4, IL-5, andIL-13 in multiple organs accompanied by blood eosinophilia,increased IgE titers, and hypertrophy of airway epithelia andincreased mucus production.41 IL-33 administration to the air-ways also induces a rapid influx of eosinophils and production

TABLE II. Discrete genetic signals and predicted function of SNPs associated with asthma in the IL33 and IL1RL1 gene regions

LD block RS no.

Association

reference SNP location

Predicted

function P value Tissue Source database

IL33

2 rs1342326 5, 8 59 of IL33 21 rs2381416

rs3939286

rs928413

7

4, 15

8

59 of IL3359 of IL3359 of IL33

2 rs2066362 5 Intron 1 22 rs16924159 9 Intron 1 22 rs12551256 9 Intron 1 22 rs7025417 9 Intron 1 22 rs2026991 39 of IL33 eQTL* 1.00E-24 Liver GTEx 17

IL1RL1

2 rs11693697 59 of IL1RL1 eQTL� 4.95E-04 Fat tissue GeneVar 18

1 rs1420089 12 Intron 1a Unknown

2 rs3771180

rs13431828

rs13408661

rs1041973

rs10173081

rs10197862

7

9, 11, 15

6

9, 11, 13

7

14

Proximal promoter

Exon 2 (59UTR)Intron 2

Exon 3 (nonsynonymous)

Intron 5

Intron 10

Amino acid

substitution

3 rs1420101

rs12998521

4, 10 Exon 5E (synonymous)

39 of IL1RL1

eQTL�

eQTL�

6.88E-04

1.23E-04

Primary peripheral

blood CD41 lymphocytes

Primary peripheral

blood CD41 lymphocytes

(Murphy 19)

(Murphy 19)

4 rs1946131 12 Intron 8 Unknown

2 rs1921622 10 Intron 10 Unknown

5 rs1861245

rs4988955

rs4988956

rs4988957

rs10192036

rs10204137

rs4988958

rs10192157

rs10206753

rs3755276

rs3771166

12

5, 9

5, 9

5, 9

5, 9, 16

Intron 10

Intron 10


Exon 11 (synonymous)



Exon 11 (synonymous)



59 of IL18R1Intron 2 of IL18R1

Amino acid

substitution

and eQTL�

3.05E-04

3.05E-04

3.78E-10

3.05E-04

3.05E-04

3.05E-04

3.05E-04

3.05E-04

3.05E-04

3.05E-04

Fat tissue

Fat tissue

Monocytes

Fat tissue

Fat tissue

Fat tissue

Fat tissue

Fat tissue

Fat tissue

Fat tissue

GeneVar 18

GeneVar 18

SeeQTL 20, 21

GeneVar 18

GeneVar 18

GeneVar 18

GeneVar 18

GeneVar 18

GeneVar 18

GeneVar 18

Polymorphisms with a reference in column 3 have been associated with asthma. eQTLs are depicted in italics, whereas IL1RL1 polymorphisms associated with asthma are depicted

in boldface. The Methods section in this article’s Online Repository describes Table II’s construction method.

eQTL, Expression quantitative trait locus; UTR, untranslated region.

*eQTLs associated with IL33 mRNA levels.

�eQTLs associated with IL1RL1 mRNA levels.

�eQTLs associated with IL18R1 mRNA levels.


MARCH 2013


of IL-5 and IL-13 by innate immune cells independently of IL-4and TH2 lymphocytes.35,58 This innate response characterized byhigh levels of IL-5 and IL-13 is now generally referred to as aninnate type 2 response.Inmurine models IL-33 has been found to be released promptly

after activation or damage of lung resident cells by, for instance,protease-active or glycolipid allergens35,59,60 or influenza infec-tion,61 as well as after allergen challenge in sensitized mice.23

On release, IL-33 activates an innate population of IL-5– andIL-13–producing cells, which were originally identified in mousemodels by multiple groups as ‘‘natural helper cells’’ or ‘‘nuo-cytes’’62,63 and now commonly referred to as ILC2s.64 Also, inthe lung ILC2s have been shown to be present and to respondto IL-33 and to contribute to airway inflammation34 and airwayhyperresponsiveness.33 In fact, ILC2s were found to be required

and sufficient in experimental models of allergic airway diseasefor IL-5 and IL-13 production and mucus hypersecretion on pro-tease treatment.59 Importantly, the induction of airway hyperres-ponsiveness and goblet cell hyperplasia by IL-33 has been foundto be dependent on IL-1RL1 signaling and MyD88-TIR interac-tion.65 In experimental mouse models of allergic airway inflam-mation, ILC2s were found to be a major source of IL-5 andIL-13 but not of IL-4.34 Importantly, the existence of theseIL-33–responsive innate immune cells producing large quantitiesof IL-5 and IL-13 has recently also been described in human sub-jects.57,66 Further support for a central role of the ILC2 cell pop-ulation in the pathogenesis of asthma stems from theidentification of retinoic acid–related orphan receptor a (RORA)as an asthma gene in the GABRIEL GWA study5 and the meta-analysis of the Analysis in Population-based Cohorts for Asthma

TIRTIR

IL-1RAcP

IL1-RL1a

Airway epithelial cells

DendriticCells

Allergen exposure Viral infection

TLRs

IL-1RL1bIL-1RL1b

IL-1RL1b

Naive Th cellsTh2 cells Innate immune cells:

Basophils, Mast cells

Type-2 innatelymphoid cells

Th2 differentationIL-4, IL-5, IL-13 production

enhanced degranulationIL-4, IL-5, IL-13 production

IL-5, IL-13 production

Inhibition of TLR-mediated signaling

TLR2 TLR4

TIR

NF-κB

TIR TIR TIR

IL-1RL1bMALMyD88

IL-33-induced signaling

IL-1RL1a

ERKAP-1

TIR TIR TIR TIR TIR

MALMyD88

IL-1RL1b

NF-κB

IL33IL33 IL33IL33IL33

IL-1RL1b

IL33IL33

TIR

TIR

IL33

TIR TIRTIR

sequestration of

signaling adaptor proteins

inhibition of

TLR signaling

heterotrimeric complex

formation and signalingIL-33 binding

inhibition of

IL-33 binding

IL33

IL33

TIRTIR

IL33IL33

IL33

IL33

IL33

IL33IL33

IL33 IL33 IL33

FIG 2. Biological activity of the IL-33/IL-1RL1 signaling pathway. IL-33–producing and responsive cells in the

lungand the IL-33/IL-1RL1 signaling pathway (in part basedonLingel et al26) are shown.Airway epithelial dam-

agewill induce IL-33 release. IL-33binding toa receptor complexof IL-1RL1-band IL-1RAcP initiates recruitment

of Mal or MyD88 signaling adaptor molecules, leading to activation of downstream pathways (lower insert).IL-1RL1-a canneutralize IL-33. In addition to its role in IL-33 signal transduction, IL-1RL1b can inhibit TLR signal-

ing through TIR domain–dependent sequestration of the adaptor proteins MyD88 andMal (top insert).




Traits consortium with GABRIEL data7 because RORa was re-cently shown to be critically required for the differentiation ofILC2s in vivo.67 Taken together, a central role in the pathogenesisof asthma for the IL-33/IL-1RL1 pathway, acting in part throughactivation of ILC2s, is supported by the genetic evidence stem-ming from multiple independent GWA studies identifying 3asthma genes operating in this pathway.Next to ILC2s, IL-33 also activates TH2 cells, mast cells, and

basophils, inducing the production of IL-4, as well as IL-5 andIL-13.68 For instance, IL-33 was shown to act as a chemoattrac-tant for TH2 cells,46 and activation of TH2 cells in the presenceof IL-33 greatly enhanced their production of IL-5 and IL-13 inan IL-1RL1- and MyD88-dependent but IL-4–independent fash-ion.49,58 Basophils responded to IL-33 through enhanced basaland IgE cross-linking–induced production of IL-4, IL-5, andIL-13.49,52 Additionally, mast cells responded to IL-33 with en-hanced survival and an increased basal and IgE cross-linking–in-duced production of IL-4, IL-5, and IL-13.47,69-71 Of note, unlikeILC2s, these innate cells are capable of producing IL-4 on IL-33stimulation, a response that has recently been shown to also occurin vivo in a T cell–independent fashion.72

From these in vitro and in vivo studies, a picture is emergingthat higher levels of IL-33, higher activity of IL-1RL1-dependent signal transduction, or both will result in an aggravatedinnate type 2 response mediated by ILC2s, mast cells, and baso-phils, as well as an enhanced adaptive TH2 response. The rele-vance of these observations from experimental mouse models

of asthma for human disease is underscored by studies in asth-matic patients: IL-33 protein was found to be increased in thebronchoalveolar lavage fluid and in airway epithelial cells73 andairway smooth muscle cells of asthmatic patients comparedwith those of healthy control subjects, which positively correlatedwith asthma severity.74,75 Moreover, several cell types derivedfrom allergic subjects, including mast cells, basophils, and eosin-ophils, could be matured and activated to release IL-4, IL-5, and/or IL-13 on IL-33 stimulation, which was increased comparedwith that seen in cells isolated from healthy control subjects.47,49

CONCLUSION AND FUTURE DIRECTIONSSNPswithin the IL33 and IL1RL1 genes are reproducibly found

to associate with asthma in different populations, and evidencefrom experimental models strongly supports a functional rolefor IL-33/IL-1RL1 signaling in asthma pathogenesis. Thereforewe conclude that the IL-33/IL-1RL1 axis plays a critical role inthe susceptibility for this chronic inflammatory disease. On thebasis of currently published data, multiple discrete genetic signalscan be distinguished within the IL33 and IL1RL1 loci, each ofwhich might have an independent contribution to asthma pathol-ogy. However, the complex LD structure at the IL1RL1/IL18R1locus precludes identification of causal asthma polymorphismsby genetic studies. This hurdle could possibly be tackled byperforming association studies in populations characterizedwith less LD, such as the African (American) population,76 or


MARCH 2013


in well-powered meta-analyses with conditional analyses to in-vestigate independence of SNP effects on asthma. Finally,whereas GWA studies typically focus on prevalent variants andare able to explain only a small part of the asthma heritability,it has been shown that asthma susceptibility genes might also har-bor rare variants with potential large effects on gene function.77

Rare variants are therefore thought to explain a part of the ‘‘miss-ing heritability’’ of asthma.78 Hence resequencing of IL33 andIL1RL1 within the context of large-scale sequencing projectsmight answer the question of whether these genes indeed harborrare variants, thereby allowing the analysis of their contribution toasthma susceptibility.In addition to association studies, functional studies focused on

a single SNP or a complete haplotype could also shed light on thebiological relevance of the different genetic signals located withinthe IL-33/IL-1RL1 axis for asthma pathophysiology. One suchstudy performed by Shimizu et al19 revealed that SNPs locatedwithin the distal promoter region of IL1RL1 contribute to alteredIL1RL1 transcription levels. Clearly, more work needs to be donein this area to fully appreciate the relevance of the diversity ofIL33 and IL1RL1 genetic signals in the context of asthmapathogenesis.The recently identified ILC2 is a critical IL-33–responsive

cellular intermediate in the pathogenesis of asthma, whichcontributes to the asthma phenotype through the production ofIL-5 and IL-13. The role of the ILC2s is further supported by theidentification of RORA, encoding the transcription factor criti-cally required for ILC2 differentiation, as another asthma GWAstudy gene.5,7 The relevance of the innate ILC2s, next to the adap-tive and allergen-specific TH2 cells, for the pathogenesis ofasthma will be subject of intense research in the coming years.This research will also need to dissect the relative contributionsof ILC2s and other effector cells of the immune system, such asbasophils and mast cells, to the IL-33/IL-1RL1–driven responses.Such data will likely guide the rational design of novel interven-tions for this chronic inflammatory disease. Moreover, identifica-tion of causal asthma variants and unraveling of the functionalrelevance of these genetic signals in the appropriate cells and tis-sues might lead to a future prospect of personalized therapeuticintervention based on individual genetic risk factors. This mightbe beneficial to understand the clinical variety of asthma pheno-types and offer directions for a rational personalized interventionstrategy.

We thank Dr Mel�en for providing additional details on his studies.

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MARCH 2013

865.e1 GROTENBOER ET AL

METHODS

Methods used for assembling Table IPolymorphisms located in the IL33 and IL1RL1 genes reported to have a

significant association with asthma were included in the table by using the cri-

terion of significance as defined in each study. SNPs retrieved from candidate

studies, genome-wide association studies, and meta-analyses are presented

(see references in the table).

Methods used for assembling Table IITable II includes the reported asthma-associated SNPs from Table I classi-

fied according to LD blocks, as defined and depicted in Fig 1, A and B, based

on the LD structure found in the CEU population (HapMap release 28, phase

I–, II–, and III–merged genotypes and frequencies, based on National Center

for Biotechnology Information B36 assembly).E1 An analysis of publically

available expression quantitative trait locus databases was performed that in-

cluded datasets from the Pritchard Laboratory,E2 GeneVar,E3-E6 and Ge-

Tex,E2,E7 and expression data were retrieved for SNPs from

Caucasians.E2-E9 A search up to 100 kb 59 of the genes IL33, IL1RL1, and

IL18R1 was performed, considering an expression quantitative trait locus

with a P value of less than .001 as significant.

Methods used for defining the LD plots of Fig 1LD of the polymorphisms located within the IL33

region (chromosome 9) and the IL1RL1-IL18R1 region

(chromosome 2). Structural information of the genomic region of

IL33 and IL1RL1, as well as information regarding mRNA transcript variants,

was derived from the National Center for Biotechnology Information Refer-

ence Sequence NC_000009.11 and NC000002.11, respectively (Homo sapi-

ens, GRCh37.p5 Primary Assembly, available at http://www.ncbi.nlm.nih.

gov). LD plots were constructed with HaploView (version 4.2) software,

with SNP genotyping data downloaded from the international HapMap Project

(available at http://hapmap.ncbi.nlm.nih.gov/index.html.en)E1 for the

CEU population. HapMap release 28 (phase I–, II–, and III–merged genotypes

and frequencies) was used and areas {chr9:6170,000..6260,000} and

{chr2:102280,000..102370,000} were downloaded for IL33 and IL1RL1, re-

spectively. For reasons of clarity, a limited number of 40 SNPs was included

within each LD plot. SNP selection was done by prioritizing asthma-

associated SNPs from Table I (main text) while faithfully reporting the under-

lying LD pattern of the loci. Subsequently, LD blocks were defined by using an

r2 value of 0.8 or greater with at least 1 asthma-associated SNP as a cut-off to

include SNPs in a certain LD block.

Methods used for defining the LD plots of Figs E1

and E2We determined the extent to which LD patterns with asthma-associated

SNPs in the IL33 and IL1RL1 genomic regions differ among distinct popu-

lations worldwide. To this end, SNP genotype data were downloaded for

populations of African origin (ASW and YRI), Asian origin (CHB), and

Latin American origin (MEX) from the international HapMap Project,E1 re-

lease 27 (phase I–, II–, and III–merged genotypes and frequencies) was used,

and area {chr9:6170,000..6260,000} was selected for IL33, whereas area

{chr2:102280,000..102370,000} was selected for IL1RL1. SNP genotype

data of IL33 and IL1RL1 were analyzed by using HaploView (version 4.2)

software. LD plots were constructed by including the selected asthma-

associated SNPs shown in Fig 1 (main body text) from the CEU reference

population. SNPs with missing genotypes were excluded from the LD plots.

Subsequently, for each of the analyzed populations, the LD pattern for the

asthma-associated SNPs (see Tables I and II and the main text) was

compared with that reported in the CEU population by using an r2 value

of 0.8 or greater as a cutoff value for each SNP to be considered part of

an LD block.

Interpretation of Figs E1 and E2For Fig E1, the LD pattern of the IL33 genomic region of several popula-

tions was compared with the LD blocks defined in the CEU population. LD

block 1, as observed in the CEU population, could be broken down into inde-

pendent signals in the ASW, YRI, and CHB populations but not in the MEX

population. This resulted in the identification of multiple independent SNPs

in these ethnic backgrounds: SNPs rs3939286 (ASW population), rs928413

(ASW and CHB populations), and rs2381416 (YRI and CHB populations)

constituted independent signals with an LD of less than 0.8 (r2), SNPs that

have been associated with asthma in several white populations (Table II). In

addition, LD block 2, as observed in the CEU ethnic background, are all inde-

pendent SNPs in the YRI population, including asthma-associated SNP

rs7025417. For the asthma-associated SNPs that were independent signals

in the CEU ethnic background, some SNPs (eg, rs16924159 and

rs12551256 in the YRI or rs1342326 and rs2066362 in the MEX) now form

a new LD block, making their individual contributions harder to dissect in

those ethnic backgrounds.

For Fig E2, the LD pattern of asthma-associated SNPs of the IL1RL1/

IL18R1 genomic region of several distinct populations was compared with

the LD blocks defined in the CEU population. For all 4 populations studied,

some differences in LD pattern were apparent with regard to the asthma-

associated SNPs, although overall, (unlike the IL33 gene), the LD pattern

was remarkably similar in all ethnic backgrounds, with a high degree of LD

throughout the locus. However, it is worth noting that the nonsynonymous

SNP rs1041973 (LD block 2 in the CEU, see Tables I and II) represents an in-

dependent SNPwithin all 4 studied populations (ASW, YRI, CHB, andMEX),

an SNP that Wu et alE10 associated with asthma in the Mexican population.

Furthermore, an additional asthma-associated SNP (rs1861246, see also

Table I) that is not present in the HapMap dataset (release 27) of the other pop-

ulations is present in the CHB population and is in LD with block 3 SNPs.

Overall, functional experiments are required to further establish true causal

SNPs in the discussed areas, whereby it should be noted that not only the LD

pattern of SNPs can differ among populations but also the relationship with

asthma can prove distinct.

http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov

http://hapmap.ncbi.nlm.nih.gov/index.html.en

http://www.ncbi.nlm.nih.gov/gtex/GTEX2/gtex.cgi

http://www.ncbi.nlm.nih.gov/gtex/GTEX2/gtex.cgi

rs14

1242

6

Ars1342326

rs18

8890

9

rs99

2969

rs3939286

rs928413

rs2066362

rs16

9241

44

rs22

1046

4

rs47

4084

0

rs16

9241

61

rs12551256

rs70

3325

8

rs7025417

rs47

4217

0

rs10

9755

16

rs13

3038

3

rs10

9755

19

rs70

4792

1

rs10

4827

4

rs20

2699

1

19

48

27

42

20

7

1

1

10

10

0

6

0

3

1

6

7

7

7

1

39

0

15

5

9

5

4

5

0

6

6

1

0

4

0

1

1

1

0

45

76

49

0

0

8

13

5

0

11

3

9

0

0

3

3

3

2

63

46

0

1

3

2

3

2

2

4

13

1

0

0

0

0

16

73

0

0

5

14

4

1

9

1

6

2

2

2

2

2

8

1

0

0

2

6

0

1

0

1

0

0

0

0

0

1

3

12

2

3

5

0

6

4

1

12

9

9

9

4

4

13

0

75

17

3

31

2

14

13

13

13

13

32

12

5

13

78

2

22

8

10

10

10

18

4

18

2

25

29

0

13

10

10

10

5

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3

16

1

5

2

2

2

2

5

23

0

41

0

12

11

11

11

18

19

57

19

20

25

25

25

3

10

22

13

14

14

14

33

8

4

5

5

5

15

42

36

36

36

1

80

80

80

0

0

0

0

FIG E1. LD of the IL33 genomic region for the ASW, YRI, CHB, and MEX populations. A, ASW population.

B, YRI population. C, CHB population. D, MEX population. Boldface, Asthma-associated SNPs; underlined,expression quantitative trait loci.



GROTENBOER ET AL 865.e2

rs19

2999

6

B

rs47

4216

6

rs14

1242

6

rs

13

42

32

6

rs

23

81

41

6

rs18

8890

9

rs13

2840

60

rs99

2969

rs

39

39

28

6

rs

92

84

13

rs

20

66

36

2

rs16

9241

44

rs11

7944

19

rs22

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rs10

4358

16

rs10

9754

97

rs47

4084

0

rs

16

92

41

59

rs16

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rs

12

55

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56

rs70

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rs70

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rs13

75

rs

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41

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0

rs70

1957

5

rs10

9755

16

rs13

3038

3

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2999

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1357

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19

rs14

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1

rs70

4792

1

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4827

4

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8153

97

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15

48

37

20

39

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14

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0

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7

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16

16

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16

16

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34

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5

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22

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24

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61

8

34

15

14

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28

20

26

26

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0

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61

9

34

15

15

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21

26

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0

16

61

9

34

15

15

30

28

21

26

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6

0

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53

39

9

10

10

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65

6

6

15

24

5

23

6

5

15

15

13

14

14

4

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7

9

9

8

36

4

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22

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31

27

28

28

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96

72

72

8

69

69

2

0

76

78

8

75

75

0

0

20

96

96

6

0

19

96

96

6

0

20

20

35

43

7

0

7

0

13

FIG E1. (Continued)


MARCH 2013


rs19

2999

6

C

rs47

4216

6

rs14

1242

6

rs

13

42

32

6

rs

23

81

41

6

rs18

8890

9

rs13

2840

60

rs99

2969

rs

39

39

28

6

rs

92

84

13

rs

20

66

36

2

rs16

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44

rs11

7944

19

rs22

1046

4

rs10

4358

16

rs10

9754

97

rs47

4084

0

rs

16

92

41

59

rs16

9241

61

rs

12

55

12

56

rs70

3325

8

rs70

3472

0

rs13

75

rs

70

25

41

7

rs47

4217

0

rs70

1957

5

rs10

9755

16

rs13

3038

3

rs19

2999

2

rs11

1357

3

rs10

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19

rs14

1242

1

rs70

4792

1

rs10

4827

4

rs10

8153

97

rs20

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1

82

82

23

38

38

38

41

0

0

0

0

0

2

4

0

0

1

0

0

0

0

0

0

0

0

0

2

0

0

0

48

31

31

31

33

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0

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65

65

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76

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79

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66

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57

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73

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69

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57

55

50

50

14

22

18

17

18

18

20

18

19

14

14

13

16

13

13

14

13

82

78

81

78

78

78

78

75

71

71

56

58

53

56

47

53

95

95

95

95

95

95

91

86

86

68

71

67

68

59

63

86

82

82

63

65

60

63

63

66

85

85

86

66

67

63

64

64

69

86

82

82

65

65

62

63

63

67

86

82

82

63

65

60

63

63

66

86

81

81

63

65

59

63

63

65

86

82

82

64

65

61

63

63

67

95

95

75

78

75

75

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53

79

82

79

79

69

56

80

82

80

79

69

58

90

77

90

75

88

73

90

77

76

FIG E1. (Continued)




rs1342326

D

rs18

8890

9

rs99

2969

rs3939286

rs928413

rs2066362

rs16

9241

44

rs22

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4

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0

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8

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0

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16

rs13

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3

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19

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1

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4

rs20

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1

73

54

61

61

86

25

0

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22

23

22

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74

74

62

28

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26

25

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26

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32

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39

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25

59

59

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16

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9

9

10

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48

16

14

13

14

14

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43

53

54

53

55

12

59

17

18

17

18

18

6

59

20

17

16

17

17

0

16

17

17

17

18

6

84

83

84

83

6

8

9

8

8

FIG E1. (Continued)


MARCH 2013


rs11

6936

97

Ars

1168

5424

rs12

7121

35

rs

14

20

08

9

rs12

7121

41

rs

37

711

80

rs

13

43

18

28

rs

10

41

97

3

rs

14

20

10

1

rs37

7117

5

rs12

7121

42

rs

19

46

13

1

rs

19

21

62

2

rs

10

19

78

62

rs49

8895

5

rs

10

20

41

37

rs

10

19

21

57

rs

10

20

67

53

rs37

5527

6

rs13

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8

rs

37

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66

1

1

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3

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10

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1

3

10

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2

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75

34

36

13

13

44

13

13

13

7

4

13

3

2

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1

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44

24

12

12

42

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12

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1

12

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0

0

0

0

0

0

28

10

10

33

17

10

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5

9

10

0

0

0

0

1

1

1

37

37

12

0

37

0

1

12

37

3

4

4

4

2

2

2

31

33

33

10

17

23

21

21

21

24

24

24

31

33

33

10

17

23

21

21

21

24

24

24

1

31

1

16

17

31

11

10

10

10

4

4

4

33

31

23

33

28

28

28

28

34

34

34

33

10

17

23

21

21

21

24

24

24

31

23

33

28

28

28

28

34

34

34

8

10

8

7

7

7

5

5

5

17

55

56

56

56

48

48

48

23

21

21

21

24

24

24

87

87

87

87

87

87

87

87

87

87

87

87

FIG E2. LD of the IL1RL1 genomic region for the ASW, YRI, CHB, and MEX populations. A, ASW population.

B, YRI population. C, CHB population. D, MEX population. Boldface, Asthma-associated SNPs; underlined,expression quantitative trait loci.




rs11

6854

24

B

rs12

7121

35

rs11

1239

18

rs11

6904

43

rs

14

20

08

9

rs14

2008

8

rs67

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4

rs19

9746

7

rs12

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41

rs

37

711

80

rs

13

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18

28

rs13

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69

rs13

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61

rs

10

41

97

3

rs

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81

rs37

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9

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5

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13

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rs65

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9

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93

rs13

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55

rs17

6963

76

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42

rs

19

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62

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19

78

62

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61

24

5

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5

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57

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67

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66

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15

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50

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49

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56

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6

6

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13

25

10

9

9

9

9

2

3

13

13

13

1

1

3

2

2

2

2

2

31

38

2

2

2

2

13

2

2

2

2

2

5

4

5

5

5

17

18

23

23

23

23

6

5

19

19

19

21

21

21

21

21

10

8

23

23

23

7

6

91

91

91

7

7

91

91

91

7

7

91

91

91

7

7

91

91

91

7

7

91

91

91

8

8

8

7

7

7

FIG E2. (Continued)


MARCH 2013


rs11

6936

97

C

rs11

6854

24

rs12

7121

35

rs11

1239

18

rs10

1826

39

rs11

6904

43

rs1420089

rs14

2008

8

rs11

1239

20

rs67

0684

4

rs19

9746

7

rs12

7121

41

rs3771180

rs13431828

rs13

4085

69

rs13

4086

61

rs1041973

rs10173081

rs37

3212

9

rs1420101

rs37

7117

5

rs12

7121

42

rs1946131

rs65

4311

9

rs13

0289

93

rs13

0174

55

rs17

6963

76

rs12

9995

42

rs1921622

rs10197862

rs1861246

rs1861245

rs49

8895

5

rs10204137

rs10192157

rs10206753

rs12

1058

08

rs37

5527

6

rs13

6234

8

rs3771166

81

94

3

10

10

10

20

10

50

68

7

68

5

68

5

68

5

57

10

64

2

2

2

2

2

2

2

2

87

77

88

81

81

81

81

81

3

9

9

9

17

9

37

52

6

53

4

53

4

53

4

58

9

73

14

14

14

14

14

14

14

14

3

10

10

10

18

10

54

69

6

69

4

69

4

69

4

57

10

72

4

4

4

4

4

4

4

4

84

84

84

84

88

3

8

9

9

15

9

46

67

5

62

3

59

3

59

3

57

8

69

4

3

3

3

3

3

3

3

3

9

9

9

17

9

56

70

6

70

4

71

4

71

4

59

9

73

4

3

3

3

3

3

3

3

3

10

10

10

19

10

52

68

7

68

4

69

4

69

4

57

10

66

2

2

2

2

2

2

2

2

3

10

10

10

19

10

52

68

7

68

4

69

4

69

4

57

10

66

2

2

2

2

2

2

2

2

3

10

10

10

19

10

52

68

7

68

4

69

4

69

4

57

10

66

2

2

2

2

2

2

2

2

3

10

10

10

19

10

52

68

7

68

4

69

4

69

4

57

10

66

2

2

2

2

2

2

2

2

3

10

10

10

19

10

50

68

7

68

4

68

4

68

4

57

10

65

2

2

2

2

2

2

2

2

87

87

87

43

87

5

8

64

8

0

8

0

8

0

9

87

0

53

53

53

53

53

53

53

53

52

5

7

73

7

0

7

0

7

0

8

0

63

63

63

63

63

63

63

63

53

4

7

73

7

0

6

0

6

0

8

0

63

63

63

63

63

63

63

63

53

4

7

73

7

0

6

0

6

0

8

0

63

63

63

63

63

63

63

63

53

9

0

38

0

25

0

25

0

25

1

52

3

52

52

52

52

52

52

52

52

4

7

73

7

0

6

0

6

0

8

0

63

63

63

63

63

63

63

63

73

3

74

2

75

2

75

2

61

5

52

7

8

8

8

8

8

8

8

5

7

7

7

82

7

68

11

11

11

11

11

11

11

11

5

0

5

0

5

0

6

73

0

46

46

46

46

46

46

46

46

7

7

7

82

7

68

11

11

11

11

11

11

11

11

7

7

5

0

4

3

3

3

3

3

3

3

3

7

7

82

7

69

10

11

11

11

11

11

11

11

7

5

0

4

3

3

3

3

3

3

3

3

7

82

7

69

10

11

11

11

11

11

11

11

5

0

4

3

3

3

3

3

3

3

3

8

83

0

0

0

0

0

0

0

0

0

63

63

63

63

63

63

63

63

1

1

1

1

1

1

1

1

FIG E2. (Continued)




rs11

6936

97D

rs11

6854

24

rs12

7121

35

rs1420089

rs12

7121

41

rs3771180

rs13431828

rs1041973

rs1420101

rs37

7117

5

rs12

7121

42

rs1946131

rs1921622

rs10197862

rs49

8895

5

rs10204137

rs10192157

rs10206753

rs13

6234

8

rs3771166

25

28

1

26

17

22

12

6

23

6

0

2

20

4

4

4

4

3

4

95

4

21

22

35

56

22

56

9

37

18

2

2

2

2

2

2

4

95

17

18

31

56

18

56

9

37

15

0

0

0

0

0

0

4

1

1

1

2

1

2

0

3

1

19

19

19

19

19

19

22

24

36

54

24

54

9

35

20

2

2

2

2

2

2

83

59

3

83

3

1

4

75

32

32

32

32

32

32

66

5

5

0

6

91

42

42

42

42

42

42

0

66

0

25

0

59

28

28

28

28

27

28

5

17

76

5

12

11

11

11

11

11

5

0

6

91

42

42

42

42

42

42

17

76

5

12

11

11

11

11

11

13

0

2

2

2

2

2

2

7

0

0

0

0

0

0

46

46

46

46

46

46

FIG E2. (Continued)


MARCH 2013


Decoding asthma: Translating genetic variation in IL33 … · Decoding asthma: Translating genetic variation in IL33 and ... the gene encoding the transcription factor critical for

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