Family-based association analysis implicates IL-4 in susceptibility to Kawasaki disease

Family-based association analysis implicates IL-4 insusceptibility to Kawasaki disease

JC Burns1, C Shimizu1, H Shike1, JW Newburger2, RP Sundel2, AL Baker2, T Matsubara3,Y Ishikawa3, VA Brophy4, S Cheng4, MA Grow4, LL Steiner4, N Kono5, and RM Cantor51 Department of Pediatrics-0830, University of California San Diego, School of Medicine, La Jolla,CA, USA2 Departments of Cardiology and Pediatrics, Boston Children’s Hospital, Boston, MA, USA3 Department of Pediatrics, Yamaguchi University, Yamaguchi, Japan4 Department of Human Genetics, Roche Molecular Systems, Alameda, CA, USA5 Departments of Human Genetics and Pediatrics, David Geffen School of Medicine at Universityof California Los Angeles, Los Angeles, CA, USA

AbstractSeveral compelling lines of evidence suggest an important influence of genetic variation insusceptibility to Kawasaki disease (KD), an acute vasculitis that causes coronary artery aneurysmsin children. We performed a family-based genotyping study to test for association between KDand 58 genes involved in cardiovascular disease and inflammation. By analysis of a cohort of 209KD trios using the transmission disequilibrium test, we documented the asymmetric transmissionof five alleles including the interleukin-4 (IL-4) C(−589)T allele (P = 0.03). Asymmetrictransmission of the IL-4 C(−589)T was replicated in a second, independent cohort of 60 trios (P =0.05, combined P = 0.002). Haplotypes of alleles in IL-4, colony-stimulating factor 2 (CSF2),IL-13, and transcription factor 7 (TCF7), all located in the interleukin gene cluster on 5q31, werealso asymmetrically transmitted. The reported associations of KD with atopic dermatitis andallergy, elevated serum IgE levels, eosinophilia, and increased circulating numbers of monocyte/macrophages expressing the low-affinity IgE receptor (FCεR2) may be related to effects of IL-4.Thus, the largest family-based genotyping study of KD patients to date suggests that geneticvariation in the IL-4 gene, or regions linked to IL-4, plays an important role in KD pathogenesisand disease susceptibility.

Keywordspediatrics; vasculitis; coronary artery aneurysm; Th2 immune response; transmissiondisequilibrium test; polymorphism

Correspondence: Dr JC Burns, Dept of Pediatrics-0830, University of California San Diego, School of Medicine, 9500 Gilman Dr., LaJolla, CA 92093-0830, USA. [email protected] Database InformationOnline Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nkh.gov/Omim/.Supplementary information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene).

NIH Public AccessAuthor ManuscriptGenes Immun. Author manuscript; available in PMC 2010 July 28.

Published in final edited form as:Genes Immun. 2005 August ; 6(5): 438–444. doi:10.1038/sj.gene.6364225.

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http://www.ncbi.nlm.nkh.gov/Omim/

http://www.nature.com/gene

IntroductionKawasaki disease (KD) is an acute, self-limited vasculitis that is now the leading cause ofacquired heart disease in children in the US and Japan. 1,2 The vascular inflammation resultsin permanent damage to coronary arteries with the development of aneurysms in up to 25%of untreated children.3 Since the original description over 30 years ago by TomisakuKawasaki, a pediatrician in Japan,4,5 investigators have searched unsuccessfully for acausative infectious agent.6 Several lines of evidence point to an important role of geneticvariation in KD susceptibility. For example, while KD has been reported in most ethnicgroups, it is over-represented among Asians and Asian/American populations and is 1.5times more common in males.7–9 In Hawaii, the annual incidence for Japanese-Americans is145/100 000 children <5 years of age, which is similar to the incidence for Japanese livingin Japan and approximately 10-fold higher than the rate for Caucasian Americans.10,11

Asians in San Diego County, where active surveillance for KD has been performed since1994, have a 2.7-fold increased risk as compared to all other ethnic groups.12 In Japan,siblings of an index case have a 10-fold increased risk of KD compared to the generalpopulation.13 In addition, the incidence of KD is two-fold higher in Japanese parents ofchildren with KD.14 Genetic association studies in KD have been performed in smallcohorts, but none have been replicated to date.15–20 A recent study used a genome-widescan in 82 Japanese KD sibling pairs and identified a polymorphism in the CD40 ligand(TNFSF5 [MIM300386]) gene on Xq26 that was associated with aneurysm formation inmale infants with KD.21

To search for genes that influence susceptibility to KD, we evaluated genetic variation incandidate genes known to be involved in inflammation and cardiovascular disease inchildren diagnosed with KD and their families (Figure 1 and Supplemental Table 1). Wechose a candidate-gene association study, rather than a genome-wide scan, because of thesmall number of families with multiple affected members and strong biologic reasons tochoose specific genes as candidates. Given that KD affects multiple ethnic groups withpotentially different risk allele frequencies, we chose a family-based study design of affectedtrios analyzed by the transmission disequilibrium test (TDT).22 Our analysis ofpolymorphisms in 58 candidate genes (Supplemental Table 1) in KD children and theirparents is the most comprehensive, family-based association study of KD patients reportedto date. We present evidence that genetic variation in interleukin-4 (IL-4) (IL4[MIM147780]) is implicated in susceptibility to KD. Further investigation of this gene maylead to new insights into KD pathogenesis.

ResultsAll 95 polymorphisms in 58 genes were in Hardy–Weinberg equilibrium in parents and KDcases in Cohorts 1 and 2 (data not shown). Differential transmission was observed for fivepolymorphic alleles in Cohort 1 at a significance level of 0.05 or less (Table 1). These fivealleles were from five different genes, which encode the following proteins (i) paraoxonase1 (PON1 [MIM168820]), an enzyme involved in lipid peroxidation, (ii) G-protein-coupledreceptor 2-like (GRK4 [MIM137026]), an intracellular kinase that inactivates severalmembers of the G-protein-coupled 7 transmembrane-spanning receptor family, (iii) IL-4, acytokine implicated in the T-helper (Th2) immune response, (iv) Transforming growthfactor-β (TGFB [MIM190180]), a pleiotropic growth factor associated with acuteinflammation, and (v) group-specific component for vitamin D binding (GC [MIM139200]),a precursor for a potent macrophage-activating factor. Of these five alleles, only the C alleleof the IL-4 promoter polymorphism C(−589) T was again preferentially transmitted inCohort 2 (P = 0.05). An analysis of this allele in the 269 trios combined from both cohortsresulted in a significance level of 0.002. Owing to the consistent difference in KD incidence

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between males and females (male predominance 1.5 : 1.0), we stratified the sample of 269trios by the sex of the affected child and reran the TDT. The trend was the same for bothsexes: 39 vs 24 (P<0.04) for girls and 63 vs 41 (P<0.02) for boys.

Polymorphisms in the genes for colony-stimulating factor 2 (CSF2 [MIM138960]), IL-13(IL13 [MIM147683]), and transcription factor 7 (TCF7 [MIM189908]), all located on 5q31(Figure 2), were among those genotyped in both cohorts. Pair-wise linkage disequilibrium(LD) between these loci and IL-4 revealed that none of these polymorphisms was incomplete LD with IL4 C(−589)T (Table 2). Although there was evidence for asymmetrictransmission of particular haplotypes, the significance levels were not greater than for theasymmetric transmission of the IL-4 C(−589)T allele alone (Table 3), suggesting that thesehaplotypes are not more specific risk factors for KD. As the IL-4RA (IL4R [MIM147781])gene is on chromosome 16p12 and encodes the α-chain of the heterodimeric receptor forboth IL-4 and IL-13, we also tested for gene–gene interactions between IL4 C(−589)T andthree IL-4RA alleles (Ile50Val, Ser478Pro, and Gln551Arg) in Cohort 1. No significantinteractions were identified in the 145 non-Hispanic Caucasian KD trios from Cohorts 1 and2 (data not shown).

The distribution of the IL-4 alleles in 121 KD patients with normal coronary arteries and 127KD patients with dilatation or aneurysms in both cohorts was not significantly different (P =0.09), suggesting no association of the IL-4 C(−589)T polymorphism with coronary arteryoutcome in KD (Table 4).

DiscussionOur study implicates IL-4 or regions linked to IL-4 in KD susceptibility but not coronaryartery outcome. The IL-4 gene is located together with IL-13, IL-9 (IL9 [MIM146931]), andIL-5 (IL5 [MIM147850]) on chromosome 5q31, which form an interleukin gene cluster.23

The association of IL-4 C(−589) with increased KD susceptibility raises interestingquestions about the role of IL-4 and Th 2-cell response in this enigmatic disease.

IL-4 is produced by activated CD4 +-Tcells, mast cells, and basophils, and affects manydifferent immunoregulatory pathways.24 IL-4 is most prominently involved in thedifferentiation of naïve Th cells to Th2 effector cells, which in turn secrete IL-4, IL-5, IL-6,IL-10, and IL-13. Several intriguing correlates between IL-4 and features of KD are worthyof mention. Elevated IL-4 levels have been reported in the serum of acute KD patients.25

Intracellular cytokine staining of peripheral blood mono-nuclear cells from KD patients hassuggested a Th1/Th2 imbalance with a predominance of the Th2 phenotype during the acutestage.26 IL-4 also regulates the expression of CD23 (low-affinity IgE receptor, FcRεII),which is expressed as an activation antigen on the surface of monocyte/macrophages, Bcells, platelets, and eosino-phils.27 Increased expression of CD23 on B cells and monocyte/macrophages has been demonstrated in acute KD.28,29 Levels of soluble CD23 are alsoincreased during acute KD.30 IL-4 also upregulates vascular cell adhesion molecule-1(VCAM-1), which is a cell surface glycoprotein expressed by cytokine-activatedendothelium that mediates the adhesion of monocytes and lymphocytes. Soluble VCAM-1levels are elevated in acute KD and may participate in the process of vascular injury.31–33

Evidence for linkage of IL-4 C(−589)T to elevated serum IgE levels, predisposition toasthma, atopic dermatitis, allergy, and severity of respiratory syncytial virus infection hasbeen reported.34–39 Serum IgE levels are elevated in acute KD28,40–42 as well as in infantilepolyarteritis nodosa, a condition thought to be synonymous with fatal KD.43,44 Theprevalence of allergy and atopic dermatitis is increased in KD patients compared to age- andethnicity-matched controls, again suggesting a link to IL-4.45–47 Thus, many features of the

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immune response in acute KD are consistent with the effects of IL-4 and a dominant Th2response, adding support to the current finding of the association of an IL-4 polymorphismwith susceptibility to KD.

In our two cohorts, the IL-4 T(−589) allele was preferentially not transmitted to offspringwith KD. The IL-4 T(−589) allele has been associated with increased promoter activity byreporter gene expression assays in a single T-cell line in vitro.48 However, in vitro assayswith cell lines may not accurately reflect the effect of a polymorphism in vivo, which isinfluenced by complex local environments and the products of other genes. Many morepolymorphisms have been described in the IL-4 gene and other genes in the interleukincluster of 5q3134 and a complex haplotype structure described.23 Analysis of haplotypesincluding IL-4 IL-13, TCF7 and CSF2 in our cohort was not more informative than the IL-4C(−589)T alone. Genotyping and haplotype transmission analysis of additional polymorphicalleles in our KD cohort may further clarify the contribution of IL-4 to KD susceptibility.

As a pilot study involving only 269 children with KD, our trial has certain limitations. Ourrelatively limited number of subjects prevented stratification by ethnicity, and forced us toeliminate analysis of alleles that are rare in non-Hispanic Caucasians. Association of adisease with an allelic variation may be detectable only in certain ethnic groups and could bemissed in an analysis performed without ethnic stratification. Thus, future studies in a largerKD cohort should include stratification by ethnicity.

In a complex disease such as KD, multiple genes are expected to influence diseasesusceptibility and outcome. The finding of an association of genetic variation in the IL-4gene and KD susceptibility is the first step toward understanding these complex geneticinfluences. Replication of these findings should be sought in additional, independentcohorts. A more focused examination of additional genes involved in the IL-4 signalingpathway may yield further insight into KD susceptibility.

Materials and methodsSubjects

All KD patients who met 4/5 standard clinical criteria or 3/5 criteria plus coronary arteryabnormalities documented by echocardiography (Online Supplement, Table S1)49 wereentered with their biologic parents into the study after obtaining informed parental consent.The Institutional Review Boards of the participating clinical centers reviewed and approvedthis study.

The first cohort (Cohort 1) consisted of 209 trios, while Cohort 2 consisted of anindependent sample of 60 trios. A total of 220 families (170 in Cohort 1 and 50 in Cohort 2)were enrolled at two clinical centers (Boston Children’s Hospital and Children’s HospitalSan Diego) at the time of hospitalization for acute KD or during a subsequent clinicevaluation. In all, 49 trios from across the United States and Canada (39 in Cohort 1 and 10in Cohort 2) who contacted the Kawasaki Disease Research Program at the University ofCalifornia San Diego were also enrolled after review of their medical histories.

Demographic and clinical data including self-reported ethnicity were collected on allsubjects (Online supplement). Of the 209 children in Cohort 1, 64.1% were male and theracial/ethnic distribution was as follows: 55.0% non-Hispanic Caucasian, 17.7% HispanicCaucasian, 13.4% Asian /Pacific Islander, 0.5% African American, and 13.4% mixed/other/unknown. In Cohort 2, 55% were male and the ethnic distribution was 50.0% non-HispanicCaucasian, 16.6% Hispanic Caucasian, 13.3% Asian /Pacific Islander, 3.3% AfricanAmerican, and 16.6% mixed/other/unknown. Coronary artery status was assessed by

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measuring the internal luminal diameter by echocardiogram during the acute and subacuteillness. In Cohorts 1 and 2, 121 patients were classified as normal and 127 patients wereclassified as either dilated (>2 and <3 s.d. above the mean for body surface area50) oraneurysmal (focal dilatation >3 s.d.). Data were not available from some patients.

Candidate gene and polymorphism selectionAs KD is characterized by immune-mediated damage to the blood vessel wall, we selected76 genes that had been previously implicated in the pathogenesis of cardiovascular andinflammatory diseases and encoded proteins involved in lipid metabolism, autonomicregulation, cell adhesion, extracellular matrix remodeling, cell signaling, platelet activation,immune response, and inflammation (Figure 1, Online supplemental Table S2). The 144polymorphisms were selected either because they had been previously associated withclinical phenotypes in cardiac or immune-mediated diseases in the published literature orbecause they were in strong LD with such polymorphisms. As noted below (Study Designand Genetic Analysis), 49 infrequent polymorphisms were not analyzed further for diseaseassociation due to lack of statistical power.

GenotypingDetailed methods for DNA preparation are presented in the Online supplement. Briefly,DNA was extracted from either 3 ml of blood16 or from 10 ml of mouthwash containingshed buccal cells.51 These methods yielded approximately 25–75 μg and 10–200 μg ofDNA, respectively. An allele-specific amplification and detection system was developed byRoche Molecular Systems (Alameda, CA, USA),52,53 and the details are presented in theOnline Supplement. Briefly, genomic regions containing polymorphic alleles were amplifiedby multiplex polymerase chain reaction (PCR) using 50 ng of DNA and biotinylated primersets. PCR products were hybridized to a linear array of sequence-specific oligo-nucleotidesimmobilized on nylon strips and detected by colorimetric methods. Genotypes were readusing software developed at Roche Molecular Systems and confirmed by visual inspectionof the strips.

Data quality controlDNA samples with known genotypes were included as controls in each experimental run.Approximately 10% of the total study population was regenotyped and all originalgenotyping results were confirmed. On the five strips used for genotyping each individual,there were nine polymorphisms duplicated on different nylon strips. Data were examined foragreement between each pair of duplicate alleles and all results were consistent. Mendelianinheritance was assessed using the Pedcheck software54 and three families who did notfollow Mendelian inheritance at multiple markers were eliminated from the data set leaving209 families in Cohort 1 and 60 families in Cohort 2 for analysis. Each polymorphism wastested for Hardy–Weinberg Equilibrium separately for each ethnicity in parents and childrenusing the Mendel software.55

Study design and genetic analysesGiven the potential for Type I statistical errors when multiple candidate genes and alleles aretested and the desire to retain sufficient statistical power to detect associations with a sampleof this size, a two-stage design was employed. In the first hypothesis-generating stage(Cohort 1), 49 alleles with a frequency estimate of less than 15% in non-HispanicCaucasians were eliminated from further analysis, as there was insufficient power to detectdifferential allele transmission with a cohort of 209 trios. The remaining 95 polymorphismsin 58 genes were analyzed as described below. The level of significance was set at 0.05,which could result in observing five alleles as a false-positive test result. In the second

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hypothesis-testing stage, five polymorphisms identified as differentially transmitted inCohort 1 were genotyped and analyzed in Cohort 2 using the same methods. Analysis ofIL-4 C(−589)T transmission was also repeated on the combined sample of 269 trios fromCohorts 1 and 2.

The TDT22 was applied to 209 trios (Cohort 1) and 60 trios (Cohort 2) using the TDTEXsoftware version 4.6 of the SAGE package.56 This analytic method applies the McNemartest of association in paired samples22 and computes an exact P-value using a permutationtest where the alleles within the pairs are shuffled within trios. Significant preferentialtransmission of alleles from heterozygous parents to their affected children was interpretedas hypothesis-generating evidence implicating that gene in KD susceptibility. The TDTapproach allowed families from different ethnic backgrounds to be analyzed together.

After the IL-4 C(−589) allele was found to be associated with KD, we examined LD ofgenes near IL-4 on chromosome 5q31.1 that were already genotyped in Cohort 1. Pair-wiseLD with IL-4 C(−589)T was assessed separately for 28 Asian and 115 non-HispanicCaucasian trios in Cohort 1 using an EM algorithm approach as programmed in thegraphical overview of linkage disequilibrium (GOLD) software.57 None of thesepolymorphisms was in complete LD with IL4 C(−589)T, and thus, each was analyzed as ahaplotype with the IL-4 polymorphism for preferential transmission to KD children inCohort 1. The Transmit software,58 which infers haplotype frequencies using the EMalgorithm, was employed. To assess gene–gene interactions between IL-4 and the IL4receptor on Chromosome 16, an exact and doubly ordered association analysis of genotypesof KD Caucasian children in Cohort 1 was conducted using the StatExact software.59 Theassociation of the IL-4 polymorphism with coronary artery status was analyzed using acontingency table analysis of a case–control study of 103 KD patients with normal coronaryarteries (controls) and 106 KD patients with dilatation of the coronary arteries or aneurysmsin Cohort 1.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThis work was supported by grants from the National Institutes of Health, NIH-RO1HL69413 and K24HL074864(awarded to JCB) and by a Grant-in-Aid from the American Heart Association, Western Affiliate #035061Y(awarded to JCB). Some of the results of this paper were obtained by using the program package SAGE which issupported by a US Public Health Service Resource Grant (RR03655) from the National Center for ResearchResources. We thank John F Bastian for patient referral and collection of DNA samples. We also thank our clinicalnurses Ellen McGrath, and Jennifer Foley, and the army of students including David Bronstein, Jennie Buchanan,Marina Dergun, Christina Lin, Erin Miller, and Mathew Leach without whose help we could never have collectedall the DNA samples required for this study. We also thank Calvin Mano, Tracy Nguyen, and Nang Tan (RMS) fortheir support in producing the genotyping reagents, and Jeff Post (RMS) for the strip interpretation software usedfor this work.

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Burns et al. Page 9


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Figure 1.Candidate genes implicated in inflammatory and cardiovascular disease pathways werechosen for genotyping in KD trios.

Burns et al. Page 10


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Figure 2.Schematic of Chromosome 5 showing the interleukin gene cluster and location of genesincluded in genotyping (italic) and haplotype anaylsis (bold).



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Tabl

e 1

Poly

mor

phis

ms d

iffer

entia

lly tr

ansm

itted

from

par

ents

to K

D p

atie

nts i

n C

ohor

ts 1

and

2

Gen

esPo

lym

orph

ism

(alle

le 1

,po

sitio

n, a

llele

2a )

Gen

e lo

catio

nA

llele

2 fr

eque

ncy

Coh

ort 1

(n =

209

trio

s)C

ohor

t 2 (n

= 6

0 tr

ios)

Cau

casi

ans

Asi

ans

Het

eroz

ygou

s par

ents

bA

llele

2 P

:N

PcP-

valu

eH

eter

ozyg

ous p

aren

tsA

llele

2 P

: NP

P-va

lue

PON

1G

ln19

2Arg

7q21

.30.

330.

6219

3/41

211

2:81

0.03

48/1

2023

:25

NS

GPR

K2L

Arg

65Le

u4p

16.3

0.38

0.12

179/

416

74:1

050.

0351

/118

23:2

8N

S

IL-4

C(−

589)

T5q

23–3

10.

180.

8313

7/41

856

:81

0.03

30/1

189:

210.

05

TGF-β

C(−

509)

T19

q13.

10.

320.

5418

0/41

610

8:72

0.00

955

/118

22:3

3N

S

GC

Thr4

20Ly

s4q

12–1

30.

270.

1615

1/41

689

:62

0.03

41/1

1822

:19

NS

PON

1 =

Para

oxon

ase

1; G

PRK

2L =

G-p

rote

in-c

oupl

ed re

cept

or k

inas

e 2-

like;

IL-4

= In

terle

ukin

-4; T

GF-β=

trans

form

ing

grow

th fa

ctor

-β, G

C =

grou

p-sp

ecifi

c co

mpo

nent

for v

itam

in D

bin

ding

; NS

=no

nsig

nific

ant.

a For n

onsy

nony

mou

s cod

ing

sequ

ence

pol

ymor

phis

ms,

the

amin

o-ac

id p

ositi

on is

num

bere

d fr

om th

e tra

nsla

tiona

l sta

rt si

te; f

or p

rom

oter

pol

ymor

phis

ms,

the

nucl

eotid

e po

sitio

n is

num

bere

d fr

om e

ither

the

trans

crip

tiona

l sta

rt si

te (T

GF-β)

or t

he tr

ansl

atio

nal s

tart

site

(IL-

4).

b Num

ber o

f het

eroz

ygou

s par

ents

/tota

l par

ents

.

c P : N

P, n

umbe

r of m

inor

alle

les p

asse

d : n

ot p

asse

d fr

om h

eter

ozyg

ous p

aren

t to

child

.


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Table 2

Pairwise (D′) with IL-4 C(−589)T

Polymorphism Caucasian D′ Asian D′

KD children (n =115) Parents (n = 230) KD children (n =28) Parents (n =56)

CSF2 Ile117Thr 0.36 0.13 0.68 0.62

IL-13 intron C/T 0.44 0.42 0.22 0.18

TCF7 Pro19Thr 0.23 0.18 0.0 0.0

IL = interleukin; CSF = colony-stimulating factor; TCF =transcription factor.

Parents and children with KD were analyzed separately in non-Hispanic Caucasian and Asian populations.


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Tabl

e 3

Tran

smis

sion

of h

aplo

type

s in

com

bine

d co

horts

(n =

269)

bas

ed o

n al

lele

s in

IL-4

, IL-

13, C

SF2

and

TCF7

CSF

2 Il

e117

Thr

IL-1

3 In

tron

C/T

IL-4

C-(5

89)T

TC

F7 P

ro19

Thr

P-va

lue

Pref

eren

tially

tran

smitt

ed h

aplo

type

s—

CC

—0.

03

Ile—

C—

0.03

——

CPr

o0.

002

Ile—

CPr

o0.

02

Pref

eren

tially

not

tran

smitt

ed h

aplo

type

sIle

—T

—0.

008

——

TPr

o0.

002

Ile—

TPr

o0.

02

IL =

inte

rleuk

in; C

SF =

colo

ny-s

timul

atin

g fa

ctor

; TC

F =

trans

crip

tion

fact

or.

Res

ults

are

show

n on

ly fo

r hap

loty

pes t

hat w

ere

asym

met

rical

ly tr

ansm

itted

(P<0

.05)

.


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Table 4

Distribution of IL-4 C(-589)T alleles stratified by coronary artery status in 248 KD children

Genotype CAA status

Normal Dilateda Aneurysm

CC 70 (58)b 33 (49) 38 (64)

CT 38 (31) 19 (28) 16 (27)

TT 13 (11) 16 (23) 5 (9)

Total 121 68 59

az score >2 and <3.

bn (%).


Family-based association analysis implicates IL-4 in susceptibility to Kawasaki disease

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