Online Supplement for:
Family-Based Association Analysis Implicates IL-4 in Susceptibility to
Kawasaki Disease
1
Jane C. Burns M.D , Chisato Shimizu MD1, Hiroko Shike MD1, Jane W. Newburger MD2,
Robert S. Sundel MD2, Tomoyo Matsubara MD3, Yuichi Ishikawa3, Suzanne Cheng PhD4,
Michael A. Grow4, Lori L. Steiner4, Victoria A. Brophy4, Naoko Kono MS5 Rita M. Cantor PhD5
1
Dept. of Pediatrics, University of California San Diego, School of Medicine, La Jolla, CA
Depts of Cardiology and Pediatrics, Boston Children's Hospital, Boston MA
3
Dept. of Pediatrics, Yamaguchi University, Yamaguchi, Japan
4
Dept. of Human Genetics, Roche Molecular Systems, Alameda CA
5
Depts. of Human Genetics and Pediatrics, David Geffen School of Medicine at UCLA, Los
Angeles, CA
2
TABLE OF CONTENTS
1. Human subjects
Table S1. Diagnostic criteria for KD
2. Clinical data
3. DNA sample preparation
3. Allele-specific amplification
4. Allele-specific detection
1. Human subjects
All patients with KD or a history of KD who meet 4/5 standard clinical criteria (Table S1) or 3/5
criteria plus coronary artery abnormalities documented by echocardiography and for whom both
biologic parents agreed to donate DNA samples were entered into the study after obtaining
informed consent.
2. Clinical data
Clinical data including gender, self-reported ethnicity and race, date of birth, date of disease
onset, response to intravenous gamma globulin therapy, and coronary artery status were recorded
for all subjects. All echocardiograms during the two months following disease onset were
recorded as either normal (all three vessels within 2 standard deviations of the mean internal
diameter for body surface area of patient according to the Newburger criteria, see de Zorzi et al.,
1998) or abnormal.
3. DNA sample preparation
For children <6 yrs., 3 ml of blood was collected into tubes containing EDTA and DNA was
extracted using the using the Wizard Genomic DNA extraction kit (Promega) as previously
published (Quasney et al., 2001). This procedure routinely yielded 25-75 μg of PCR-quality
genomic DNA. For parents and KD children over the age of 6 yrs., 10 ml of Scope mouthwash
was be used to collect shed buccal cells for DNA extraction (Heath et al., 2001). The yield was
between 10-200 μg of PCR-amplifiable genomic DNA.
4. PCR amplification
Polymorphic alleles were amplified using a multiplex polymerase chain reaction (PCR)
developed by Roche Molecular Systems(Alameda CA)52,53. A 50-ul PCR mixture contained 50
ng of sample DNA and multiple sets of biotinylated primers at optimal concentrations as
supplied by Roche Molecular Systems. A total of 4 primer pools were used to amplify all 144
polymorphic loci using AmpliTaq® Gold. Samples were amplified in a Perkin-Elmer GeneAmp
9600 thermocycler using the following thermal cycling profiles for the cardiovascular disease
allele assays (CVD A and B) and inflammation allele assay (INF) (Table S2):
 initial hold of 94oC for 7min ("hot start" protocol)
 33 cycles of 2-step PCR (95oC for 15 sec and 60oC for 1min for CVD assay) or 34 cycles of
3-step PCR (95oC for 15 sec, 61oC for 30 sec and 72oC for 1 min for INF assay)
 final extension step of 68oC for 5min for CVD assay and 72oC for 7min for INF assay
 hold at 10oC until analysis.
5. Allele-specific detection
We used nylon strips printed with specific oligonucleotide sequences and a colorimetric
detection method provided Roche Molecular Systems. Four different strips were used to detect
the corresponding multiplex PCR products generated with four primer pools, CVD: A-C and
INF. Strips were incubated with 3 ml of hybridization buffer. For the CVD assay the
hybridization/wash buffer conatined 2x SSPE (0.36 M NaCl, 0.02 M Na2HPO4, 2 mM EDTA,
adjusted to pH 7.4 with NaOH), 0.5% SDS. For the INF assay, the hybridization buffer contained
3 x SSPE and 0.5% SDS. Multiplex PCR product (20 l) was denatured with 20 l of 1.6%
NaOH and added to the wells with corresponding strips and hybridization buffer.Hybridization
was performed at 53 oC for CVD A and C, 52.5 oC for CVD B, and 55 oC for INF assay using a
water bath rotating at 50-60 rpm (Hot Shaker Plus; Bellco). After 15-20 minutes, strips were
quickly rinsed in hybridization/wash buffer for the CVD assay or wash buffer (1.5 x SSPE, 0.5%
SDS) for INF assay. Next, strips were incubated with streptavidin-horseradish peroxidase
conjugate (SA-HRP) for 5 min. in the same buffer used for hybridization. The strips were
washed with stringent wash buffer (2x SSPE, 0.5% SDS), and incubated for 12 min. The washed
strips were equilibrated in 50 mM Na citrate at room temperature on a rotating (50-60 rpm)
platform, then agitated in color development reagent for 8-10 min at room temperature. Color
development reagent was a mixture of substrate A (0.01% H2O2 in Na citrate solution) and
substrate B (0.1% 3,3’,5,5’-tetramethylbenzidine in 40% dimethylformamide) in a ratio of 5:1.
Developed strips were rinsed with distilled water, aligned on a flat surface next to a guide
identifying the allele detected by each probe line, and scanned into a computer database. Scanned
images were converted to Excel file data either by computer program (Roche Molecular System)
or by reading the results manually.
Table S1. Diagnostic criteria for KD
The diagnosis of KD is considered confirmed by the presence of fever of at least 5 days
duration and four of the remaining five criteria, and if the illness cannot be explained by
some other known disease process.
Bilateral conjunctival injection
Changes of the mucous membranes of the upper respiratory tract: injected pharynx, injected,
fissured lips, strawberry tongue
Changes of the peripheral extremities: peripheral edema, peripheral erythema, periungual
desquamation
Polymorphous rash
Cervical adenopathy
Table S2. Complete list of gene and alleles analyzed in Cohort 1. Highlighted genes/alleles have
an estimated frequency of <15% in non-Hispanic Caucasians.
Symbol Gene name
Polymorphism dbSNP rs#
Location
ACE
A(-262)T
17q23
17q23
17q23
4p16.3
5q31-q32
5q31-q32
5q31-q32
8p11
1q42
3q24
3q24
3q24
11q23.3
11q23.3
2p24
2p24
2p24
2p24
11q23.3
11q23.3
11q23.3
11q23.3
11q23.3
11q23.3
19q13
19q13
19p13.3-p13.2
9q32-q34
21q22.3
17q21.1-q21.2
17q21.1-q21.2
3p21
3p21.3
3p21
3p21
5q22-32
16q21
16q21
16q21
16q21
16q21
16q21
16q21
5q31.1
2q33
2q33
10q11.1
11p11.1
1q23
13q34
13q34
13q34
13q34
5q33
6p25
6p25
4q28
4q12-q13
4q12-q13
12p13
17pter
17pter
17pter
4p16
4p16
4p16
19p13.2
19p13.2
2q12-q21
2q14
2q14
Angiotensin I-converting enzyme
Thr776Thr (A/G)
ADD1
ADRB2
Adducin 1; alpha adducin
Beta-2 adrenergic receptor
ADRB3
AGT
AGTR1
Beta-3-adrenergic receptor
Angiotensin I
Angiotensin receptor 1
APOA4
Apolipoprotein A-IV
APOB
Apolipoprotein B
APOC3
Apolipoprotein C-III
APOE
Apolipoprotein E
C3
C5
CBS
CCL11
Complement component 3
Complement component 5
Cystathionine beta-synthase
chemokine (C-C motif) ligand 11
CCR2
CCR3
CCR5
Chemokine receptor 2
Chemokine receptor 3
Chemokine receptor 5
CD14
CETP
CD14 antigen
Cholesteryl ester transfer protein
A20320G
Gly460Trp
Arg16Gly
Gln27Glu
Thr164Ile
Trp64Arg
Met235Thr
G(-535)A
T(-153)C
A1166C
Thr347Ser
Gln360His
Thr71Ile
Pro2712Leu
Arg3500Gln
Glu4154Lys
C(-641)A
C(-482)T
T(-455)C
C1100T
C3175G
T3206G
Cys112Arg
Arg158Cys
Arg102Gly
Ile802Val
Ile278Thr
G(-1328)A
Ala23Thr
Val62Ile
Pro39Leu
G(-2459)A
32bp-ins/del
C(-260)T
C(-631)A
C(-629)A
TaqIB +/- (G/A)
Ile405Val
Asp442Gly
intron 14 G(+1)A
intron 14 (+3)Tins
CSF2
CTLA4
CXCL12
F2
F5
F7
Colony stimulating factor 2
Cytotoxic T-lymphocyte-associated protein 4
Ile117Thr
C(-318)T
Thr17Ala
Chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) G880A
Coagulation factor II or prothrombin
G20210A
Coagulation factor V
Arg506Gln
Coagulation factor VII
G(-402)A
G(-401)T
-323 10-bp del/ins
F12
F13A1
FGB
GC
GNB3
GP1BA
GRK4
ICAM1
IL1A
IL1B
Arg353Gln
C(-46)T
Val35Leu
Pro564Leu
Fibrinogen, beta polypeptide chain
G(-455)A
Group specific component for vitamin D binding protein Asp416Glu
Lys420Thr
Guanine nucleotide-binding protein, beta-3
C825T
Glycoprotein Ib (platelet), alpha polypeptide
G(-1691)T
T(-5)C
Thr145Met
G protein-coupled receptor kinase 4
Arg65Leu
Ala142Val
Ala486Val
Intercellular adhesion molecule 1
Lys56Met
Gly241Arg
Interleukin 1 alpha
T(-889)C
Interleukin 1 beta
C(-1418)T
Coagulation factor XII
Coagulation factor XIII, A1 polypeptide
Phe105Phe (C/T)
4291
4343
4363
4961
1042713
1042714
1800888
4994
699
1492078
275653
5186
675
5110
1367117
676210
5742904
1042031
2542052
2854117
2854116
4520
5128
4225
429358
7412
2230199
17611
5742905
4795895
3744508
1799864
5742906
1799987
333
2569190
1800776
1800775
708272
5882
2303790
5742907
5742908
25882
5742909
231775
1801157
1799963
6025
762637
7981123
5742910
6046
1801020
5985
5982
1800790
7041
4588
5443
4069688
2243093
6065
2960306
1024323
1129292
5491
1799969
1800587
16944
1143634
IL4
IL4R
Interleukin 4
Interleukin 4 receptor
C(-589)T
Ile75Val
Ser503Pro
Gln576Arg
IL5RA
Interleukin 5 receptor
G(-80)A
IL6
Interleukin 6
G(-572)C
G(-174)C
IL9
Interleukin 9
Thr113Met
IL10
Interleukin 10
C(-571)A
IL13
Interleukin 13
IntronC/T
ITGA2
Integrin, alpha-2
G873A
ITGB3
Integrin, beta-3
Leu33Pro
LDLR
Low density lipoprotein receptor
NcoI+/- (A/G)
LIPC
Lipase, hepatic
C(-480)T
LPA
Lipoprotein, Lp(a)
C93T
G121A
LPL
Lipoprotein lipase
T(-93)G
Asp9Asn
Asn291Ser
Ser447Term
LTA
Lymphotoxin alpha
Thr26Asn
Intron A/G
LTC4S
Leukotriene C4 synthase
A(-444)C
MMP1
Matrix metalloproteinase 1 (interstitial collagenase)
1G(-1607)2G
MMP2
Matrix metalloproteinase 2 (gelatinase A)
C(-1306)T
Matrix metalloproteinase 3 (stromelysin 1, progelatinase) 5A(-1171)6A
MMP3
Lys45Glu
MMP7
Matrix metalloproteinase 7 (matrilysin, uterine)
A(-181)G
C(-153)T
MMP9
Matrix metalloproteinase 9 (gelatinase B)
Arg279Gln
Arg668Gln
C(+6)T
MMP12
Matrix metalloproteinase 12 (macrophage elastase)
A(-82)G
Asn122Ser
Membrane-spanning 4-domains, subfamily A, member 2 (FCER1B) Glu237Gly
MS4A2
MTHFR
5,10-methylenetetrahydrofolate reductase
C677T
Asp346Asp (C/T)
NOS2A
Nitric oxide synthase 2 alpha
NOS3
Nitric oxide synthase 3
A(-922)G
C(-690)T
Glu298Asp
NPPA
Natriuretic peptide precursor A
G664A
T2238C
PAI1
Plasminogen activator inhibitor 1
G5(-675)G4
G11053T
PDE4D
Phosophodiesterase 4D, cAMP-specific
SNP222*
SNP220*
SNP219*
SNP199*
SNP175*
SNP148*
PON1
Paraoxonase 1
C(-108)T
Met55Leu
Gln192Arg
PON2
Paraoxonase 2
Ser311Cys
PPARG
Peroxisome proliferator-activated receptor-gamma
Pro12Ala
REN
Renin
G(-1111)A
C2646T
SCGB1A1 Secretoglobin, family 1A, member 1 (uteroglobin)
G(+38)A
SCNN1A Sodium channel, nonvoltage-gated 1 alpha
Trp493Arg
Ala663Thr
SELE
Selectin E
Ser128Arg
Leu554Phe
SELP
Selectin P
Ser330Asn
Val640Leu
TCF7
Transcription factor 7
Pro19Thr
TGFB1
Beta-1 transforming growth factor
C(-509)T
TNF
Tumor necrosis factor
G(-376)A
G(-308)A
G(-244)A
G(-238)A
VCAM1
Vascular cell adhesion molecule 1
T(-1594)C
Vitamin D (1,25- dihydroxyvitamin D3) receptor
VDR
Met1Thr
Intron 8 G/A
2243250
1805010
1805015
1801275
2290608
1800796
1800795
2069885
1800872
1295686
1062535
5918
5742911
1800588
1652503
1800769
1800590
1801177
268
328
1041981
909253
730012
1799750
243865
3025058
679620
11568818
11568819
2664538
2274756
20544
2276109
652438
569108
1801133
1137933
1800779
3918226
1799983
5063
5065
1799768
7242
27727
1988803
6450512
27547
27171
154025
705379
3202100
662
7493
1801282
6681776
2368564
3741240
5742912
2228576
5361
5355
6131
6133
5742913
1800469
1800750
1800629
673
361525
1041163
2228570
1544410
5q23-q31
16p11.2-p12.1
16p11.2-p12.1
16p11.2-p12.1
3p26-p24
7p21-p15
7p21-p15
5q31-q35
1q31-q32
5q31
5q11.2
17q21
19p13.2
15q21
6q26
6q26
8p22
8p22
8p22
8p22
6p21.3
6p21.3
5q35
11q22
16q13
11q22
11q22
11q21
11q21
20q11
20q11
20q11
11q22
11q22
11q13
1p36
17q11.2-q12
7q36
7q36
7q36
1p36.2
1p36.2
7q22
7q22
5q12
5q12
5q12
5q12
5q12
5q12
7q21
7q21
7q21
7q21
3p25
1q32
1q32
11q11-qter
12p13
12p13
1q23
1q23
1q21-24
1q21-24
5q31
19q13.1
6p21.3
6p21.3
6p21.3
6p21.3
1p32-p31
12q13.1
12q13.1
*Gretarsdottir S, Thorleifsson G, Reynisdottir ST, Manolescu A, Jonsdottir S, Jonsdottir T, Gudmundsdottir T, et al.
(2003) The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nat Genet 35:131-138