as a novel susceptibility gene for early childhood …...1 supplementary information a genome-wide...

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Supplementary information

A genome-wide association study identifies Cadherin Related Family

Member 3 (CDHR3) as a novel susceptibility gene for early childhood

asthma with severe exacerbations

Klaus Bønnelykke, Patrick Sleiman, Kasper Nielsen, Eskil Kreiner-Møller, Josep M Mercader,

Danielle Belgrave, Herman T den Dekker, Anders Husby, Astrid Sevelsted, Grissel Faura-

Tellez, Li Juel, Lavinia Paternoster, Richard Flaaten, Anne Mølgaard, David E Smart, Philip F

Thomsen, Morten A Rasmussen, Silvia Bonàs-Guarch, Claus Holst, Ellen A Nohr, Rachita Yadav,

Michael E March, Thomas Blicher, Peter M Lackie, Vincent WV Jaddoe, Angela Simpson, John

W Holloway, Liesbeth Duijts, Adnan Custovic, Donna E Davies, David Torrents , Ramneek

Gupta, Mads V Hollegaard, David M Hougaard, Hakon Hakonarsson, Hans Bisgaard

Contents:

Supplementary figures

Supplementary tables

Supplementary Note

References

Nature Genetics: doi:10.1038/ng.2830

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Supplementary Figure 1. Flow chart for case selection

1,743,869 children

Born in Denmark between January

1st 1982 and December 31st 2008

511 children

Exluded due to:

Competing diagnosis during

hospitalization (150)

Other chronic disease (225)

Birth weight < 2,500 g (201)

Gestational age < week 36

(176)

(Groupings are not mutually

exclusive)

2,179 children

Fulfilling inclusion criterium of >=

2 hospitalization in the age 2-6 yr

1,564 children

1,173 children

Final case population for GWAS

1,547 children

363 children

Excluded during quality

control of genotype data and

clustering with HapMap CEU

17 children

With insufficient DNA for

genotyping

104 children

Without available blood spot

sample in the biobank

1,668 children


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Supplementary Figure 2. Probability plot for the discovery analysis


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Supplementary Figure 3. Regional association plots (LocusZoom)1 from the discovery

analysis for the 5 genome-wide significant loci. The upper panel shows associations for

genotyped SNPs and the lower panel shows associations after regional imputation.

Rs2305480


5

Rs928413


6

Rs6871536


7

Rs1558641


8

Rs6967330


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Supplementary Figure 4. Forest plot of the association with asthma for the CDHR3 top-SNP

(rs6967330) in the discovery and replication studies.


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Supplementary Figure 5. Cumulative risk of acute severe asthma exacerbation (defined by

acute admission to hospital/emergency department and/or acute need of corticosteroid

treatment) during the first 6 years of life stratified on CDHR3 (rs6967330) genotypes. Data

are from combined analysis of the COPSAC2000 and MAAS birth cohorts (replication cohorts).

The analysis includes a total of 1,091 children of whom 163 had a severe exacerbation. The

genotype distribution was as follows: AA = 30 individuals, AG = 312 individuals, GG = 749

individuals. The P value for the association between genotype and risk of exacerbation was

0.006 (Cox regression analysis using an additive genetic model).


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Supplementary Figure 6. Regional association plot for the CDHR3-region in the GABRIEL

replication-study (childhood-onset stratum). Moffat MF et al. N Engl J Med 2010;125:328-35.


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Supplementary Figure 7. Regional association plot for the CDHR3-region in the replication-

subsample of 980 individuals of non-European descent (Generation R sub-sample).


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Supplementary Figure 8. CDHR3 protein expression in experimental models of the CDHR3 variant (rs6967330 / C529Y). Plasmids encoding wild-type and C529Y mutant CDHR3, as well as empty pBabe+CMV-Puro as a control, were transiently transfected into 293T cells. Cells were analyzed 48 hours after transfection. Data are representative of 3 independent experiments (transfections). (A) Transfected 293T cells were stained for Flag expression at the cell surface, followed by PE-conjugated goat-anti-mouse secondary antibody. Cells were fixed, permeabilized, and stained again with the anti-Flag antibody, followed by Alexa-647-conjugated goat-anti-mouse secondary antibody. Data were acquired by flow cytometry and gated by FSC and SSC for intact-cell-sized events. Data presented represent the surface and intracellular staining observed in gated events. The percentages shown in the top two quadrants represent the percent of gated events that are positive for intracellular staining but negative for surface staining (upper left quadrant) and the percentage of gated events positive for both intracellular and surface expression (upper right quadrant). (B) Transfected cells were lysed, and whole cell lysates were separated by SDS-PAGE under reducing or non-reducing conditions. Proteins were transferred to PVDF, and western blotted for Flag.


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Supplementary Figure 9. Representative images of HEK293T-cells transfected with either

C529Y-mutant or WT-plasmid. Cells were immunostained for extracellular CDHR3 (green),

then fixed and afterwards permeabilized and immunostained again, this time for intracellular

CDHR3 (red). Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) (blue).

C529Y-mutant

C529Y-WT


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Supplementary Figure 10. Immunohistochemical localization of CDHR3 (1in 25) in human

lung tissue epithelial cells embedded in GMA. (A) Adult lung tissue, (B) Foetal lung tissue (8.5

- 9 weeks) and (C) Negative control (adult lung). Brown: CDHR3 immunolabeling; Blue: nuclei.

Scale bar represents 50µm.


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Supplementary Table 1. Baseline characteristics of cases from the COPSACexacerbation (discovery) cohort compared to the general population born in the same period (Jan 1st 1982 - Dec 31st 2008) Cases General population N N (%) / Mean ± SD N N (%) / Mean ± SD

Gender, Male 1,173 791 (66 %) 1,744,805 895,766 (51%) Mothers smoking during pregnancy 743 226 (32.0 %) 1,136,578 261,960 (15.0 %) Older siblings at time of birth, 0 / 1 / 2 / 3+ 1,173 48% / 36% / 13% / 3% 1,744,805 45% / 37% / 14% / 5% Delivery, caesarean section 1,173 152 (13 %) 1,696,644 265,521 (16 %) Gestational age, weeks 1,173 39.7 ± 1.4 1,682,955 39.4 ± 2.0 Birth weight, kg 1,173 3.6 ± 0.5 1,684,278 3.5 ± 0.6


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Supplementary Table 2. Characterization of symptom burden in cases from the COPSACexacerbation (discovery) cohort.

N Median (IQR)

N (%)

Number of asthma hospitalizations 0-6 years 1135 3 (2 - 5) Number of asthma and/or acute bronchitis hospitalizations 0-6 years 1135 4 (3 - 6) Age at first asthma hospitalization 1173 2.63 (2.01 - 3.59) Age at first asthma or acute bronchitis hospitalization 1173 2.27 (1.31 - 3.30) First asthma hospitalization before age 1 year 1173 86 (7.3 %) First asthma or acute bronchitis hospitalization before age 1 year 1173 198 (16.9 %) First asthma hospitalization before age 2 years 1173 286 (24.4 %) First asthma or acute bronchitis hospitalization before age 2 years 1173 456 (38.9 %) First asthma hospitalization before age 3 years 1173 709 (60.4 %) First asthma or acute bronchitis hospitalization before age 3 years 1173 805 (68.6 %) Any diagnosis of allergy, allergic rhinitis, allergic asthma, allergic conjunctivitis or atopic eczema, 0-6 years

1135 664 (58.2 %)


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Supplementary Table 3. Validation of results from the discovery analysis by re-genotyping of the top-SNP from the 5 genome-

wide significant loci in cases. Validation genotyping was performed by single SNP genotyping using the PCR KASPar genotyping

system (KBiosciences, Hoddesdon, UK). Association tests were performed using the 2511 control individuals from the discovery

analysis.

Discovery genotyping

Validation genotyping

Comparison

Chr. SNP/effect allele

Position (bp)

Nearest gene

Effect allele freq. in controls

Effect allele freq. in cases OR [95% CI] P-value

Effect allele freq. in cases OR [95% CI] P-value

N with successful genotyping in both systems

Concordance rate

17 rs2305480/G 35315722 GSDMB 0.54 0.73 2.28 [2.04-2.55]

1.3E-48 0.73 2.33 [2.09-2.59]

3.9E-52 1119 0.990

9 rs928413/G 6203387 IL-33 0.25 0.34 1.50 [1.34-1.67]

4.2E-13 0.34 1.51 [1.36-1.68]

6.4E-14 1091 0.993

5 rs6871536/C 131997773 RAD50 0.20 0.27 1.44 [1.28-1.62]

1.8E-09 0.26 1.40 [1.24-1.57]

2.5E-08 1107 0.995

2 rs1558641/G 102132297 IL1R1 0.84 0.89 1.56 [1.34-1.81]

6.6E-09 0.89 1.51 [1.30-1.74]

4.2E-08 1102 0.998

7 rs6967330/A 105445687 CDHR3 0.17 0.22 1.45 [1.28-1.66]

1.4E-08 0.22 1.45 [1.28-1.64]

7.7E-09 1110 0.992


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Supplementary Table 4. Validation of results from the discovery analysis by use of an alternative control group genotyped on a

different platform. Association analysis for the 5 genome-wide significant loci were repeated using 2,688 control individuals from

the Welcome Trust Case Control Consortium 2 (WTCCC2)-project genotyped on the Affymetrix 6.0 platform. Association tests were

performed using cases from the discovery analysis.

Illumina 610 controls (Discovery)

Affymetrix 6.0 controls (Validation)

Chr. SNP/effect allele

Position (bp)

Nearest gene

Effect allele freq. in cases

Effect allele freq. in controls OR [95% CI] P-value

Effect allele freq. in controls OR [95% CI] P-value

17 rs2305480/G 35315722 GSDMB 0.73 0.54 2.28 [2.04-2.55] 1.3E-48 0.52 2.52 [2.27-2.80] 7.0E-68

9 rs928413/G 6203387 IL-33 0.34 0.25 1.50 [1.34-1.67] 4.2E-13 0.26 1.44 [1.30-1.60] 8.6E-12

5 rs6871536/C 131997773 RAD50 0.27 0.20 1.44 [1.28-1.62] 1.8E-09 0.19 1.59 [1.42-1.78] 1.6E-15

2 rs1558641/G 102132297 IL1R1 0.89 0.84 1.56 [1.34-1.81] 6.6E-09 0.84 1.54 [1.33-1.79] 4.3E-09

7 rs6967330/A 105445687 CDHR3 0.22 0.17 1.45 [1.28-1.66] 1.4E-08 0.17 1.42 [1.25-1.60] 1.5E-08


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Supplementary Table 5. Case and control definitions and numbers for asthma phenotypes in the replication studies.

Asthma ever Asthma/wheeze before 3 years

Asthma exacerbations

Study Phenotype definition N cases/ controls Phenotype definition

N cases/ controls Phenotype definition

N cases/ controls

Replication 1

GABRIEL (Moffatt et al. N Engl J Med 2010; 125:328-35) Childhood onset subsample including 19 different studies

Cases: asthma ever before 16 years Controls: no asthma

6,783/7,720 - - - -

Replication 2 (CDHR3 top SNP only)

COPSAC2000 Cases: asthma ever before 6 years Controls: no asthma before 6 years

63/282 Cases: asthma/recurrent wheeze ever before 3 years Controls: no asthma/recurrent wheeze before 3 years

76/240 1) Cases: hospitalization due to asthma age 0-6 years Controls: no asthma hospitalization 0-6 years 2) Cases: severe exacerbation requiring hospitalization, oral steroids and/or high dose inhaled steroids. Controls: no severe exacerbation

35/330 56/309

MAAS

Cases: asthma ever before 6 years Controls: no asthma before 6 years


294/568 1) Cases: hospitalization due to asthma age 0-6 years Controls: no asthma hospitalization 0-6 years 2) Cases: severe exacerbation requiring hospitalization / visit emergency department and/or oral steroids. Controls: no severe exacerbation

57/669 107/619

Generation R European sub-sample


100/1,752 Cases: asthma/recurrent wheeze ever before 3 years Controls: no asthma/recurrent wheeze before 3 years

226/1,395 - -

Generation R non-European sub-sample



148/647 - -


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Supplementary Table 6. (A-B) Association of the CDHR3 variant (rs6967330) with exacerbations, asthma and other atopic traits

in the COPSAC2000, MAAS and Generation R replication cohorts. A) Survival analyses of asthma exacerbations. B) Logistic regression

analyses of asthma and atopic traits.

A)

COPSAC2000 MAAS Combined analysis Combined analysis adjusted for study site

Meta-analysis (fixed- effects model)

Heterogeneity between studies

HR [95%CI]; P value

HR [95%CI]; P value

N cases/controls HR [95%CI) P value P value P value P value

Asthma hospitalization (0-6 years)

1.61 [0.96-2.72]; 0.07

1.74 [1.10-2.74]; 0.02

92/999 1.70 [1.20-2.40]

0.002

0.002

0.005

0.83

Asthma exacerbation (0-6 years)

1.57 [1.04-2.37]; 0.03

1.35 [0.95-1.92]; 0.09

163/928 1.44 [1.10-1.89]

0.006

0.007

7.1 E-04

0.58


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B)

COPSAC2000 MAAS Generation R European sub-sample

Generation R non-European sub-sample

Generation R combined

Meta-analysis COPSAC2000+MAAS+ Generation R

OR [95%CI]; P value

OR [95%CI]; P value

OR [95%CI]; P value

OR [95%CI]; P value

OR [95%CI]; P value OR [95%CI] P value

Asthma ever 0-6 years 1.39 [0.86-2.24]; 0.17

1.47 [1.11-1.96]; 0.007

0.88 [0.58-1.33]; 0.37

2.02 [1.14-2.11]; 6.4E-04

1.33 [1.00-1.76]; 0.05

1.40 [1.16-1.67]

3.2 E-04

Asthma/recurrent wheeze ever 0-3 years

1.22 [0.78-1.92]; 0.37

1.32 [1.01-1.73]; 0.04

1.24 [0.94-1.62]; 0.12

1.55 [1.14-2.11]; 0.004

1.39 [1.14-1.70]; 8.8E-04

1.35 [1.16-1.57]

7.5 E-05

Asthma with exacerbation (0-6 years) Asthma without exacerbation

1.54 [0.85-3.17]; 0.15 1.32 [0.68-2.41]; 0.41

1.76 [1.16-2.66]; 0.006 1.38 [0.92-2.08]; 0.11

- -

- -

- -

1.68 [1.21-2.34] 1.36 [0.97-1.91]

0.002 0.08

Number of exacerbations (0-6 years) >=2 exacerbations 1 exacerbation

1.75 [0.94-1.42]; 0.07 1.27 [0.60-0.86]; 0.52

1.51 [0.89-2.55]; 0.12 1.35 [0.83-2.19]; 0.21

- -

- -

- -

1.60 [1.08-2.38] 1.33 [0.89-1.98]

0.02 0.16

Eczema ever 0-6 years 0.95

[0.63-1.45]; 0.81 0.97 [0.75-1.27]; 0.84

1.16 [0.99-1.37]; 0.06

1.11 [0.88-1.41]; 0.36

1.09 [0.93-1.29]; 0.28

0.93 [0.82-1.06]

0.30

Allergic sensitization by age 5/6 years

0.78 [0.50-1.24]; 0.29

1.43 [1.09-1.89]; 0.008

- - - 1.22 [0.97-1.54]

0.09*

Meta-analysis P value in bold if < 0.05

* P value calculated by a random effects model due to evidence of heterogeneity between studies (P value for interaction < 0.05)


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Supplementary Table 7. Association of the CDHR3 variant (rs6967330) with bronchial

responsivenesss and lung function in the COPSAC2000 birth cohort.

GG AG/AA Bronchial responsiveness N Median (IQR) N Median (IQR) P value

1 month of age:

PD15TcO2; µmol methacholine 228 0.35 (0.12;0.92) 96 0.23 (0.10;0.74) 0.10W

4½ years of age:

Change in sRaw after cold-air hyperventilation (%) 127 8.1 % (-4.4;18.6) 58 7.7 % (-5.6;21.2) 0.78W

6 years of age:

Change in sRaw after cold-air hyperventilation (%) 160 7.3 % (-2.5;18.1) 75 7.6 % (-4.5;24.6) 0.84W

6½ years of age:

PD20; µmol methacholine 158 1.20 (0.51;2.84) 79 0.96 (0.44;2.39) 0.47W

Lung function N Mean ± SD / Median (IQR) N

Mean ± SD / Median (IQR) P value

1 month of age:

Neonatal FEV0.5(Z-score) 249 0.045 ± 1.03 111 -0.10 ± 1.02 0.22t

Neonatal FEF50(Z-score) 247 0.001 ± 1.04 110 -0.01 ± 0.96 0.93t

4 years of age:

sRaw (kPa*s) 158 1.29 (1.10;1.48) 74 1.26 (1.08;1.39) 0.36W

6 years of age:

sRaw (kPa*s) 186 1.27 (1.11;1.45) 89 1.20 (1.08;1.39) 0.36W

7 years of age:

Calibrated FEV1 (L) 195 1.44 ± 0.18 95 1.46 ± 0.19 0.62t

W P value from Wilcoxon rank-sum test t P value from unpaired t-test


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Supplementary Table 8. Variants located within predicted regulatory regions and in LD (r2 > 0.5 in 1000 Genomes reference

panel (CEU)) with the top SNP (rs6967330) at the CDHR3 locus.

Top SNP

r2 with the top SNP

Functional annotation

Promoter histone marks

Enhancer histone marks (Cell ID, state)

DNAse (Production center, cell ID, treatment)

rs6967330 1 Non-synonymous K562,4_Strong_Enhancer . .

rs114947103 0.97 Intronic K562,4_Strong_Enhancer . .

rs10258293 0.9 Synonymous K562,5_Strong_Enhancer; GM12878,7_Weak_Enhancer

CD8.MP,6_TssD2;CD4.NP,6_TssD2;CCIP.LSMPTP,2_TssF;CD3.P,2_TssF

AWG,K562,None; AWG,Th1,None; Duke,Chorion,None; Duke,GM12891,None; UW,Adult_CD4_Th0,None; UW,Th2,None

rs75853071 0.68 Intronic GM12878,6_Weak_Enhancer;K562,6_Weak_Enhancer

. UW,GM06990,None; UW,GM12864,None; UW,GM12865,None

rs10488047 0.68 Intronic GM12878,7_Weak_Enhancer;K562,7_Weak_Enhancer

. .

rs56235770 0.91 Intronic K562,7_Weak_Enhancer . AWG,Th1,None; UW,Adult_CD4_Th0,None; UW,HA-sp,None

rs74928390 0.66 Intronic K562,7_Weak_Enhancer . .

rs140882188 0.73 Intronic H1,6_Weak_Enhancer NCC.GED2,6_TssD2;BN.AC,6_TssD2;BN.SN,6_TssD2;ST.SMUS28,2_TssF

.

rs35699146 0.6 Intronic H1,6_Weak_Enhancer NCC.GED2,6_TssD2;BN.AC,6_TssD2;ST.SMUS28,2_TssF;BN.FE1,6_TssD2;BN.HM150,6_TssD2

AWG,H1-hESC,None; Duke,H9ES,None; Duke,Osteobl,None; UW,BE2_C,None; UW,CD34+_Mobilized,None; UW,H7-hESC,None; UW,HA-sp,None; UW,HConF,None; UW,HPAF,None; UW,NT2-D1,None

rs75850640 0.68 Intronic H1,7_Weak_Enhancer . .

rs76871421 0.68 UTR-3 . . Duke,Ishikawa,Estradiol_100nM_1hr; UW,H7-hESC,None

rs6959584 0.7 UTR-3 K562,7_Weak_Enhancer . .





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Supplementary Table 9. Amino acid sequence at position 529 in the CDHR3 protein for 36

species of vertebrates including 30 species of mammals. Human seems unique in having

cysteine (C) as wild type. For 9 other non-human primates tyrosine (Y) is consistent. ‘N’ refers

to the number of animals genotyped for each species. Data are obtained from NCBI

(http://www.ncbi.nlm.nih.gov/protein/) and UniProt (http://www.uniprot.org/) databases.

Species Common name Group Order 529 N

Homo sapiens Human Mammals Primates C* -

Pan troglodytes Chimpanzee Mammals Primates Y 1

Pan paniscus Bonobo Mammals Primates Y 1

Gorilla gorilla Gorilla Mammals Primates Y 3

Pongo abelii Sumatran orangutan Mammals Primates Y 1

Nomascus leucogenys Northern white-cheeked gibbon Mammals Primates Y 1

Papio anubis Olive baboon Mammals Primates Y 1

Saimiri boliviensis Black-headed squirrel monkey Mammals Primates Y 2

Callithrix jacchus Common marmoset Mammals Primates Y 1

Otolemur garnettii Greater galago Mammals Primates Y 1

Tursiops truncatus Common bottlenose dolphin Mammals Cetacea Y 1

Orcinus orca Killer whale Mammals Cetacea Y 1

Sus scrofa Pig Mammals Artiodactyla Y 1

Ovis aries Sheep Mammals Artiodactyla Y 1

Bos taurus Domestic cow Mammals Artiodactyla Y 1

Equus caballus Horse Mammals Perissodactyla H 1

Trichechus manatus West Indian manatee Mammals Sirenia Y 1

Loxodonta africana African elephant Mammals Proboscidea H 1

Odobenus rosmarus Walrus Mammals Carnivora Y 1

Ailuropoda melanoleuca Giant panda Mammals Carnivora Y 1

Canis familiaris Dog Mammals Carnivora Y 1

Felis catus Cat Mammals Carnivora Y 1

Mus musculus House mouse Mammals Rodentia H 2

Cricetulus griseus Chinese hamster Mammals Rodentia H 1

Rattus norvegicus Brown rat Mammals Rodentia H 1

Spermophilus tridecemlineatus Thirteen-lined ground squirrel Mammals Rodentia Y 2

Cavia porcellus Guinea pig Mammals Rodentia Y 1

Monodelphis domestica Gray short-tailed opossum Mammals Didelphimorphia F 1

Sarcophilus harrisii Tasmanian devil Mammals Dasyuromorphia F 1

Ornithorhynchus anatinus Duck-billed platypus Mammals Monotremeta Y 1

Gallus gallus Chicken Birds Galliformes Y 1

Meleagris gallopavo Turkey Birds Galliformes Y 1

Taeniopygia guttata Zebra finch Birds Passeriformes Y 1

Anolis carolinensis Carolina anole Reptiles Squamata Y 1

Xenopus tropicalis Western clawed frog Amphibians Anura Y 1

Latimeria chalumnae West Indian ocean coelacanth Fish Coelacanthi- formes F 1

* Approximately 65% are homozygous for the cysteine-associated allele in European populations


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Supplementary Table 10. Age-stratified replication results for the genome-wide significant loci in the discovery analysis.

Replication results are from a previously published large-scale genome-wide association study of asthma (GABRIEL). Moffat MF et al.

N Engl J Med 2010;125:328-35.

Discovery

Replication (GABRIEL)

Childhood onset (< 16 years) Adult onset (>16 years)

Het P value (Interaction with age)

Chr. SNP Nearest gene OR [95% CI] P value

P value OR [95% CI] P value P value

17 rs2305480/G GSDMB 2.28 [2.04-2.55] 1.3E-48

1.32 [1.23-1.39] 6.5E-23 0.98 [0.91.1.06] 0.68 6.6E-10

9 rs928413/G IL-33 1.50 [1.34-1.67] 4.2E-13

1.24 [1.17-1.32] 8.7E-13 1.10 [1.01-1.19] 0.03 0.01

5 rs6871536/C RAD50 1.44 [1.28-1.62] 1.8E-09

1.17 [1.10-1.25] 7.6E-07 1.08 [0.99-1.18] 0.10 0.12

2 rs1558641/G IL1R1 1.56 [1.34-1.81] 6.6E-09

1.11 [1.04-1.19] 0.003 1.08 [0.97-1.19] 0.15 0.58

7 rs6967330/A CDHR3 1.45 [1.28-1.66] 1.4E-08

1.18 [1.10-1.27] 3.0E-06 1.05 [0.95-1.16] 0.32 0.055


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Supplementary Table 11. Association results from the current study for top SNPs from a previous large-scale genome-wide

association study of asthma (GABRIEL). Moffat MF et al. N Engl J Med 2010;125:328-35.

Previous GWAS (GABRIEL)

Current GWAS

Current GWAS Stratified on number of asthma related hospitalizations

2 3 4-5 6 or more

N=272 N=228 N=277 N=358

Gene Chr SNP/effect allele

OR [95% CI]; P value

Genotyped/ imputed






IL18R1 2 rs3771166/G 1.15 [1.10-1.20]; 3.4E-09

Imputed 1.22 [1.10-1.36]; 2.8E-04

1.14 [0.94-1.37]; 0.20

1.26 [1.02-1.54]; 0.032

1.15 [0.95-1.38]; 0.16

1.32 [1.12-1.56]; 6.3E-04

HLA-DQB1 6 rs9273349/C 1.18 [1.13-1.24]; 7.0E-14

Imputed NA [SNP not in 1000G];

NA NA NA NA

IL33 9 rs1342326/C 1.20 [1.13-1.28]; 9.2E-10

Imputed 1.53 [1.35-1.73]; 1.1E-10

1.26 [1.0-1.59]; 0.049

1.30 [1.02-1.66]; 0.037

1.66 [1.34-2.05]; 5.8E-06

1.77 [1.48-2.13]; 2.1E-09

SMAD3 15 rs744910/G 1.12 [1.09-1.16]; 3.9E-09

Imputed 1.18 [1.07-1.3]; 0.002

1.03 [0.86-1.22]; 0.77

1.32 [1.09-1.60]; 0.006

1.05 [0.88-1.25]; 0.71

1.32 [1.13-1.54]; 9.5E-04

GSDMB 17 rs2305480/G 1.18 [1.11-1.23]; 9.6E-08

Genotyped 2.33 [2.07-2.56]; 8.6E-51

1.87 [1.54-2.26]; 1.5E-10

2.24 [1.81-2.78]; 2.1E-13

2.24 [1.83-2.73]; 1.7E-15

2.72 [2.26-3.29]; 3.5E-26

GSDMA 17 rs3894194/A 1.17 [1.11-1.23]; 4.6E-09

Imputed 1.59 [1.44-1.76]; 2.6E-21

1.40 [1.17-1.67]; 1.2E-04

1.53 [1.27-1.86]; 4.3E-06

1.57 [1.31-1.87]; 2.3E-07

1.82 [1.55-2.12]; 3.5E-15

IL2RB 22 rs2284033/G 1.12 [1.08-1.16]; 1.2E-08

Imputed NA [SNP not passing imputation QC];

NA NA NA NA

Suggestive in GABRIEL

SLC22A5 5 rs2073643/T 1.11 [1.06-1.15]; 2.2E-07

Imputed 1.25 [1.13-1.38]; 1.4E-05

1.11 [0.93-1.32]; 0.32

1.26 [1.04-1.52]; 0.022

1.27 [1.06-1.51]; 0.009

1.34 [1.15-1.56]; 1.6E-04

IL13 5 rs1295686/T 1.15 [1.09-1.20]; 1.4E-07

Imputed 1.31 [1.17-1.48]; 1.7E-06

1.23 [1.0-1.52]; 0.040

1.30 [1.04-1.63]; 0.016

1.30 [1.06-1.60]; 0.005

1.39 [1.17-1.66]; 1.0E-04

RORA 15 rs11071559/C 1.18 [1.11-1.25]; 1.1E-07

Imputed 1.19 [1.02-1.39]; 0.020

1.17 [0.88-1.55]; 0.29

1.14 [0.84-1.54]; 0.33

1.22 [0.92-1.62]; 0.12

1.22 [0.95-1.56]; 0.082


29

Supplementary Note

The COPSACexacerbation cohort

Diagnoses in the hospitalization register were classified according to ICD-8 (World Helath

Organization. Manual of the International Classification of Diseases. 8th rev. Geneva,

Switzerland: World Health Organization;1967) and ICD-10 (World Health Organization.

International Classification of Diseases. 10th rev. Geneva, Switzerland: World Health

Organization;2006).

Exclusion criteria: Exclusion criteria were low birth weight (<2500 g), gestational age below

36 weeks, competing side-diagnosis during hospitalization, or any registered chronic

diagnosis considered to affect risk of asthma hospitalization.

Individuals were excluded if they had any of the following diagnoses registered at any time:

Tuberculosis: ICD-8 codes 011XX-012XX, ICD-10 codes DA15X-DA19X. Malignant neoplasmas:

ICD-8 codes 140XX-207XX, 23XXX, ICD-10 codes DC0XX-DC97X, DD0XX, DD37X-DD48X.

Chronic renal diseases: ICD-8 codes 581XX-58209, 7530X-7531X, ICD-10 codes DN03X-

DN04X, DN07X-DN09X, DQ60X-DQ61X, DQ63X. Immune deficiency and other disease of

blood: ICD-8 codes 275XX, 2880X, 282XX-283XX, ICD-10 codes, DD55X-DD59X, DD7XX-

DD8XX. Mental retardation: ICD-8 codes 310XX-315XX, 7901X, ICD-10 codes DF7XX, DR620.

Paralytic and muscular diseases: ICD-8 codes 330XX-344XX, 347XX-349XX, ICD-10 codes

DG7XX-DG9XX. Cardiovascular disease: ICD-8 codes 393XX-429XX, 746XX-747XX, ICD-10

codes DI0XX-DI79X, DI95X-DI99X, DQ2XX. Other chronic lung diseases, emphysema,

bronchiectasis: ICD-8 codes 2730X, 492XX, 506XX, 5083X, 517XX-518XX, ICD-10 codes DJ43X,

DJ47X, DJ60X-DJ84X, DJ95X, DJ980, DE84X, DP27X. Congenital malformations of the nervous

system and other CNS disease: ICD-8 codes 740XX-743XX, 7613X-7614X, ICD-10 codes

DQ0XX-DQ07X. Respiratory, esophageal and gastrointestinal malformation: ICD-8 codes

530XX, 748XX-752XX, ICD-10 codes DK20X-DK23X, DQ30X-DQ45X. Chromosomal

abnormalities: ICD-8 codes 759XX, ICD-10 codes DQ90X-DQ99X. Metabolic disorders and

fakomathosis: ICD-8 codes 270XX-273XX, 275XX-276XX, 279XX, ICD-10 codes DE70X-DE72X,

DE74X-DE80X, DE83X, DE85X, DQ85X. Diabetes: ICD-8 codes 250XX, ICD-10 codes DE10X-

DE14X.


30

Phenotypic description of cases: Cases were described with respect to age at first

hospitalization from asthma (ICD8-codes 493, ICD-10 codes J45-46) or acute bronchitis

(ICD8-codes 466X, 490X, 491X, ICD-10 codes J20X, DJ409, DJ448B) and number of such

hospitalizations from 0 to 6 years of age. For the analysis of number of hospitalizations only

children with full follow up to 6 years of age (N=1,135) were included.

Cases were also characterized with respect to a concurrent atopy-diagnosis during

hospitalization (allergy, allergic rhinitis, allergic asthma, allergic conjunctivitis or atopic

eczema: ICD-8 codes 36003, 49302, 50703, 69100, 69291, ICD-10 codes DJ450, DH101A,

DH104A, DJ30X, DK137D, DK522, DL20X, DL232, DL235, DL238C, DL239, DL500, DT784).

DNA-specimens from cases: DNA was obtained from blood spots sampled as part of the

Danish neonatal screening program and stored in the Danish Neonatal Screening Biobank,

where samples are stored since 1982.2 Two disks, each 3.2mm in diameter, were punched

from each blood spot. DNA was extracted using the Extract-N-Amp kit (Sigma-Aldrich). Each

individual sample was whole-genome amplified in triplicates, each reaction containing 10µL

of DNA, using the REPLIg kit (QIAGEN) as previously described.3 The three amplifications

were pooled and the concentration of whole-genome amplified DNA (wgaDNA) was

determined by Quant-IT PicoGreen dsDNA Reagent (Invitrogen). Prior to SNP genotyping, the

samples were normalized to 50 ng/µl.

Controls: The control population consisted of adults from Denmark, who had previously been

genome-wide genotyped as participants in the Genomics of Overweight in Young Adults

(GOYA) study.4 The control samples used here were randomly drawn from two large Danish

cohorts: the Danish National Birth Cohort (females) and the Copenhagen draft board

examinations (males). Individuals who indicated in a questionnaire to have physician-

diagnosed asthma were excluded. Controls were genotyped on the Illumina Human610-Quad

v1.0 BeadChip (545,350 SNPs) at the Centre National de Génotypage (CNG), Evry, France.

The COPSAC2000 replication cohort

Asthma was diagnosed based on the following 4 mandatory criteria: (1) recurrent episodes of

troublesome lung symptoms recorded in the daily dairy cards as five episodes within 6

months, each episode lasting at least three consecutive days; (2) symptoms typical of asthma

based on doctors interviews of the parents at the clinical research unit. Such symptoms


31

included exercise induced symptoms; prolonged nocturnal cough; recurrent cough outside

common cold; symptoms causing wakening at night; (3) need for intermittent rescue use of

inhaled β2-agonist; and (4) response to a 3-month course of inhaled corticosteroids and

relapse when stopping treatment.

Treatment Algorithm: Medical treatment for asthma followed strict predefined algorithms.

For symptom relief, parents were provided with terbutaline in a pressurized metered dose

inhaler (pMDI) with a spacer to be administered as needed. Recurrent asthmatic episodes as

classified above defined the threshold for a 3-month course of 400μg of inhaled budesonide

administered by a pMDI with a spacer. Relapsing symptoms was given as six and

subsequently twelve month treatment in children responding to such treatment. Montelukast

4mg daily (5 mg after age 5) was added to the treatment of children with recurrent episodes

despite budesonide maintenance treatment. Terbutaline as needed was substituted by

Formoterol as needed for the treatment of children with recurrent episodes despite

budesonide and montelukast maintenance treatment. Acute severe asthmatic exacerbation

was treated with oral prednisolone 1-2mg/kg per day for three days (alternatively

budesonide 1600μg per day for two weeks). No other treatment was allowed for asthmatic

symptoms.

Diary registration of asthmatic symptoms: Asthmatic symptoms were recorded by the parents

in daily diaries from 1 month until 6 years of age. Asthmatic symptoms were explained to the

parents as wheeze or whistling sounds, breathlessness or recurrent troublesome cough

severely affecting the wellbeing of the infant. Emphasis was given to symptoms from the

lower airways that were not just audible but actually affecting the sleep, the activity or the

well-being of the child. The symptoms were detailed in a book that was given to the parents

and presented on the COPSAC website. The doctors at the research unit reviewed symptom

definition and the diary entries with the parents at the 6-monthly clinical sessions during 6

years. Daily symptoms were recorded as composite dichotomized scores (yes/no) on each

day.

Spirometry in neonates: Neonatal spirometry was obtained by forced flow-volume

measurements applying the raised volume rapid thoracic compression technique. The

subjects were required not to have had symptoms of respiratory infection preceding the test.


32

The forced expiratory volume at 0.5 second, FEV0.5(neonate), and the forced expiratory flow

at 50% of vital capacity, FEF50(neonate), were estimated as previously described.5-7 Baseline

was defined as the measurements after inhalation of saline.

Bronchial responsiveness to methacholine in neonates: Methacholine chloride aerosol was

administered with a dosimeter attached to a nebulizer and inhaled from a metal spacer.

Methacholine was given in quadrupling dose steps from 0.04 to 16.67 µmol. PTcO2 was

measured continuously during the methacholine challenge by the TCM3™ from Radiometer,

Copenhagen, Denmark. Lung functions were repeated in duplicates after each dose until

FEV0.5 fell by at least 20% or the maximum dose was reached. One mg terbutaline was

administered from a pressurized metered dose inhaler via spacer at the end of the test.

Our sensitivity analyses showed neonatal-PD15(TcO2) to be more sensitive than PD

determined from spirometric indices for assessment of bronchial responsiveness. We used a

nonlinear dose-response model to determine the provocative dose estimates, which

efficiently uses the data and provides sounder dose estimates than the traditional linear

interpolation of the dose-response relationship, which is more prone to variations.6

Spirometry by age 7: Lung function was measured by spirometry in the child’s 7th year of life

using a pneumotachograph Masterscope Pneumoscreen, system 754916 spirometer (Erich

Jäeger, Würtzburg, Germany). The subjects were tested sitting, and wearing nose clips.

Spirometry was assessed from up to five technically acceptable maneuvers to obtain two

flow-volume curves with less than 0.2-L difference between the largest using the higher of the

two as the outcome. Compliance was assured by computer-animated volume driven incentive

well-known to the children and all children had trained the procedures on repeated occasions

before the study day.

Specific airway resistance (sRaw) was measured at 4 and 6 years by whole body

plethysmography.8, 9

Bronchial responsiveness at age 4 and 6 years was determined as the relative change in sRaw

after hyperventilation of cold-dry air.10


33

Allergic sensitization against common inhalant allergens (cat, dog, horse, birch, timothy grass,

mugwort, D. pteronyssinus , D. farina, and moulds) was determined at age 6 years by

measurement of serum specific IgE (ImmunoCAP assay, Pharmacia Diagnostics AB, Uppsala,

Sweden).11 Sensitization was defined as a specific IgE level >= 0.35 IU/mL for any of the tested

allergens and was analyzed as a dichotomized measurement.

Eczema: Skin lesions were described at both scheduled and acute visits according to pre-

defined morphology and localization; eczema was defined based on the Hanifin-Rajka criteria

as previously detailed.12, 13

The Generation R Study

The Generation R Study is a population-based prospective cohort study of pregnant women

and their children from fetal life onwards in Rotterdam, The Netherlands.14 All children were

born between April 2002 and January 2006, and currently followed until young adulthood. Of

all eligible children in the study area, 61% were participating in the study at birth. Cord blood

samples including DNA have been collected at birth. Information about wheezing (no; yes),

asthma (no; yes) and eczema (no; yes) was collected by questionnaires at the ages of 1 to 4,

and 6 years.15 Response rates for these questionnaires were 71%, 76%, 72%, 73%, and 68%,

respectively. Questions about wheezing were ‘Has your child had problems with a wheezing

chest during the last year [never, 1-3 times, >4 times] (age 1 to 4 years)?’ and ‘Did your child

ever suffer from chest wheezing? [never, 1-3 times, >4 times] (age 6 years)?’. Questions about

asthma were ‘Has a doctor diagnosed your child as having asthma during the past year? [yes;

no] (age 2 and 4 years)’ and ‘Was your child ever diagnosed with asthma by a doctor? [yes;

no] (age 3 and 6 years)’. Based on the last obtained questionnaire, we grouped children into

‘asthma ever before age 6 years (no; yes)’. Reported asthma at age 2, 3 and 4 years were used

to reclassify ‘asthma ever before age 6 years’ where appropriate. We then re-categorized

children in those with an asthma diagnosis before age 3 years and at 3 years or older.

Reported number of wheezing episodes at age 1 and 2 years, and age 3 to 6 years,

respectively, were used to reclassify asthma diagnosis before and at age 3 years or older into

‘asthma diagnosis or >= 3 episodes of wheezing before age 3 years (no; yes)’. Questions about

eczema were ‘Has a doctor diagnosed your child as having eczema during the past year? [yes;

no] (age 1 to 4 years)’ and ‘Was your child ever diagnosed with eczema by a doctor? [yes; no]

(6 years)’. Similarly as for asthma, we grouped children into ‘eczema ever before age 6 years


34

(no; yes)’ based on the last obtained questionnaire, and used reported eczema at age 1 to 4

years to reclassify ‘eczema ever before age 6 years’ where appropriate.

Samples were genotyped using Illumina Infinium II HumanHap610 Quad Arrays following

standard manufacturer's protocols. Intensity files were analyzed using the Beadstudio

Genotyping Module software v.3.2.32 and genotype calling based on default cluster files. Any

sample displaying call rates below 97.5%, excess of autosomal heterozygosity (F<mean-4SD),

and mismatch between called and phenotypic gender were excluded. In addition, individuals

identified as genetic outliers by the IBS clustering analysis (> 3 standard deviations away

from the HapMap CEU population mean) were considered of non-Caucasian ancestry.

Rs6967330 was a genotyped SNP in this set. For the current replication study on the

association of rs6967330 with the 2 defined asthma groups (‘asthma ever before age 6 years

(no; yes)’ and ‘asthma diagnosis or >= 3 episodes of wheezing before age 3 years (no; yes)’

3,040 children were included (females, n = 1,511 (49,7%)). Ethnicity was grouped into

Caucasians (n = 1,962 (64.5%)) and Non-Caucasians (n = 1,078 (35.5%)), based on genetic

ancestry. Based on country of birth of parents and grandparents obtained by questionnaires,

the largest Non-Caucasian ethnic groups were of Turkish (5.4%), Surinamese (4.6%), Dutch

Antillean (4.0%) Moroccan (2.9%), and Cape Verdian (2.3%) origin. We included 3,282

children (Caucasians, n = 2,081 (63.4%), Non-Caucasians, n = 1,201 (36.6%)) for the

replication analyses of rs6967330 with ‘eczema ever before age 6 years (no; yes)’. Twins were

excluded from all analyses. The study protocol was approved by the Medical Ethical

Committee of the Erasmus Medical Centre, Rotterdam (MEC 217.595/2002/20). Written

informed consent was obtained from parents of all participants.

Functional studies of the CDHR3 variant

CDHR3 protein expression in experimental models of the CDHR3 gene variant (C529Y/rs6967330) CDHR3 constructs and mutagenesis: A plasmid containing the coding sequence of human

CDHR3 in the pCR-BluntII-Topo backbone was acquired from Open Biosystems (Thermo

Scientific, Lafayette CO). Quikchange Lightning mutagenesis (Agilent Technlogies, Clara CA)

was used to insert a Flag tag into the CDHR3 coding sequence, between the predicted signal

sequence and the predicted beginning of the mature polypeptide (between the 19th and 20th

amino acid of the complete protein). The primers used for the mutagenesis were: Forward -


35

atgtcagggggagaagcaGATTACAAGGATGACGACGATAAGACCGGTctacacctaatcctctta and Reverse -

taagaggattaggtgtagACCGGTCTTATCGTCGTCATCCTTGTAATCtgcttctccccctgacat. The proper

insertion of the Flag tag was confirmed by Sanger sequencing. The C529Y mutation was

generated by a second round of Quikchange Lightning mutagenesis, using the following

primers: Forward – gctggtaactaaagtCgactAtgaaacaacccccatctata and Reverse –

tatagatgggggttgtttcaTagtcGactttagttaccagc. These primers contained the point mutation

required to mutate cysteine-529 to tyrosine, as well as a second, silent point mutation that

served to introduce a SalI site for screening purposes. The introduction of the mutations and

the integrity of the entire CDHR3 sequence were confirmed by Sanger sequencing. Finally, the

Flag-tagged wild type and C529Y mutant CDHR3 sequences were amplified by PCR and

subcloned into the pBabe+CMV-Puro vector. The PCR primers used for this amplification

were: Forward – GATCCTCGAGGCCACCatgcaggaagcaatcattctcctgg and Reverse –

GATCGCGGCCGCttactttcctgggtgtggtttggg.

CDHR3 expression, flow cytometry, and western blotting. Plasmids encoding wild type or

mutant CDHR3, or the empty pBabe+CMV-Puro vector, were transfected into 293T cells by

Fugene6 transfection (Promega, Madison WI). The 293T cells were from the American Tissue

Culture Center (ATCC), catalog number: CRL-3216. They were recently tested for

mycoplasma contamination, but were not authenticated. 48 hours after transfection, cells

were removed from their plates with 3 mM EDTA in PBS. Cells were washed with PBS, and

resuspended in PBS + 0.5% BSA + 1 mM sodium azide (PBA buffer). Cells were stained for

surface Flag expression with anti-Flag antibody (clone M2, Agilent Technologies, catalog

number 200470-21, 2 g/ml), washed twice with PBA buffer, and stained with PE-conjugated

goat-anti-mouse secondary antibody (BD Biosciences, Sparks MD, 2 g/ml). Cells were

washed in PBS, fixed in 2% paraformaldehyde, and permeabilized in PBA buffer + 0.5%

saponin. Permeabilized cells were stained again for Flag (2 g/ml), washed with PBA +

saponin, stained with Alexa-Fluor-647 conjugated goat-anti-mouse secondary antibody

(Invitrogen, Grand Island NY, 20 g/ml), and washed with PBA + saponin. Cells were

resuspended in PBS, and flow cytometry was performed using the Accuri C6 instrument

(Accuri, Ann Arbor MI). Data were analyzed in FlowJo (Tree Star, Ashland OR). For western

blot, cells expressing CDHR3 proteins were lysed in PBS + 0.5% Triton X100 supplemented

with complete protease inhibitor tablets (Roche, Madison WI). Whole cell lysates were


36

separated by SDS-PAGE, under reducing or non-reducing conditions, transferred to PVDF

membranes, and blotted for Flag (M2). Three independent tranfections of the CDHR3 cDNAs

were performed on three different days. Each experiment was processed as described. Each

experiment consisted of 1 well of 293T cells transfected with the following plasmids: Empty

Vector Control, CDHR3 Wild Type in pBabe, or CDHR3 C529Y in pBabe.

Immunoflouresence and confocal microscopy. 293T cells were grown on glass coverslips with

DMEM (Dulbecco’s modified Eagle’s medium), 3 mM glutamine, 10% heat-inactivated fetal

bovine serum (FBS) at 37° C, 5% CO2 prior to and for 2 days post-transfection with expression

constructs for FLAG-tagged CDHR3 wildtype and CDHR3 C529Y variant using TransIT 2020

reagent using standard protocol (Mirus Bio LLC). Cells were obtained and used at a low

passage from ATCC and had recently been tested for mycoplasma. Cells were incubated in

10% serum containing culture media plus primary anti-FLAG mouse antibodies (F3165,

Sigma, at dilution 1:300) for 1 hour at 37° C before being washed briefly with culture media.

Then the cells were stained with secondary rabbit anti-mouse antibodies (F0261, Daco, at

dilution 1:600) conjugated with fluorescienisothiocyanate (FITC) and incubated at 37° C for

30 minutes, then washed with culture media prior to PBS. Afterwards cells were fixed in 2%

paraformaldehyde for 15 minutes, washed with PBS and permeabilized in 0.2% Triton X-100

in PBS for 5 minutes, washed and then incubated with Cy3 conjugated mouse anti-FLAG

antibody (Cy3-labelled F3165 Sigma, at dilution 1:300). Finally cells were mounted with

ProLong Gold antifade reagent with DAPI (Invitrogen). Images were acquired using a Leica

DMI 6000-B confocal microscope (Leica Microsystems, Wetzlar DE) with 40X magnification

and processed in Photoshop (Adobe Systems). The experiments were performed in triplicates

(three independent transfections) on three different days.

Functional consequence of the CDHR3 gene variant (rs6967330) on protein expression Staining transfected cells for cell surface expression of the Flag-tagged CDHR3 proteins

revealed that the wild-type protein was expressed very poorly at the cell surface, while the

C529Y variant showed better cell surface expression (Supplementary Fig. 8A). For flow

cytometry, three independent tranfections of the CDHR3 cDNAs were performed on three

different days. Each experiment was processed as described in the methods. The percentage

of cells that showed cell surface staining was consistently higher with the mutant than with

the wild type variant (at least three times higher), even when the mutant was less well


37

transfected (as shown by the intracellular staining). We chose one of the three experiments as

representative, and used that to make the figure represented in Supplementary Fig. 8A.

Intracellular staining of the transfected cells demonstrated that wild-type and C529Y CDHR3

were expressed equally well, despite the differences in cell surface expression. Western blot

analysis of lysates of tranfected cells revealed a single band when gels were run under

reducing conditions (Supplementary Fig. 8B). Running lysates under non-reducing

conditions results in the appearance of two bands of higher molecular weight, which are likely

disulfide bonded complexes of the CDHR3 molecules with other cellular proteins. However,

there were no noticeable differences between the wild-type and C529Y proteins, indicating

that the introduced mutations did not result in differences in disulfide bond formation that

could be revealed by western blot.

The results of 3 individual experiments using immunofluorescence staining consistently

showed surface expression in the risk variant only. Data (Supplementary Fig. 9) are

representative of 3 individual experiments.

Immunohistochemical localization of CDHR3 (1in 25) in Human lung tissue

Human Adult and foetal (8.5 -9 weeks approx.) lung tissues were fixed in acetone with

protease inhibitors iodoacetamide (20 mM) and phenylmethylsulphonylfluoride (PMSF) (2

mM), stored at -20°C for 24 h and processed into water-soluble glycol methacrylate (GMA)

resin. Two-micron sections were pre-treated with a solution of 0.1% sodium azide and 0.3%

hydrogen peroxide and then blocked with culture medium both for 30 minutes. After

overnight incubation at 4°C with affinity purified rabbit CDHR3 (1:25) (Sigma-Aldrich)

including Claudin-3 (1:2000) (Zymed) as positive control and TBS as negative control. Two

hours incubation with biotinylated second stage swine α rabbit (dilution 1:1200) and then

streptavidin biotin-peroxidase complexes followed with appropriate washing with TBS in

between steps. Immunoreactivity was revealed with DAB (diaminobenzidene) and

counterstained with Mayer's haematoxylin.

Statistical analyses

Replication in a previous GWAS (GABRIEL)17: The GABRIEL data

(https://beaune.cng.fr/gabriel/gabriel_results.zip) were downloaded and summary data from

the 19 included childhood onset studies were extracted and meta-analyzed in the software


https://beaune.cng.fr/gabriel/gabriel_results.zip

38

package METAL16 and PLINK (v. 1.07.) Fixed- and Random-effects P values were calculated

and reported in line with the original report.17

COPSAC2000: Cumulative risk of age at onset of exacerbations, asthma and eczema was

illustrated with Kaplan-Meier plots and Hazard Ratios were estimated with Cox regression.

The effect on sensitization was modeled by logistic regression and the effect on quantitative

measures with students T-test or Wilcoxon as appropriate.

Acknowledgments

COPSAC The authors gratefully express their gratitude to the children and families of the COPSAC

cohort study for all their support and commitment; and we acknowledge and appreciate the

unique efforts of the COPSAC research team.

Manchester Asthma and Allergy Study (MAAS) We would like to thank the children and their parents for their continued support and

enthusiasm. We greatly appreciate the commitment they have given to the project. We would

also like to acknowledge the hard work and dedication of the study team (post-doctoral

scientists, research fellows, nurses, physiologists, technicians and clerical staff).

Generation R The Generation R Study gratefully acknowledges the contributions of the children and their

parents, the general practitioners, the hospitals and the midwives and pharmacies in

Rotterdam. We would like to thank K. Estrada, T.A. Knoch, A. Abuseiris, L.V. de Zeeuw and R.

de Graaf for their help in creating GRIMP. We thank M. Jhamai, M. Ganesh, P. Arp, M. Verkerk,

L. Herrera and M. Peters for their help in creating, managing and performing quality control

for the genetic database. Also, we thank K. Estrada and C. Medina-Gomez for their support in

the creation and analysis of imputed data.

Genomics of overweight in Young Adults (GOYA) control population The GOYA study was conducted as part of the activities of the Danish Obesity Research Centre

(DanORC, www.danorc.dk) and The MRC centre for Causal Analyses in Translational

Epidemiology (MRC CAiTE), Bristol University. The genotyping for GOYA was funded by the

Wellcome Trust (WT 084762). The female part of GOYA is a nested study within The Danish

National Birth Cohort which was established with major funding from the Danish National


39

Research Foundation. Additional support for this cohort has been obtained from the

Pharmacy Foundation, the Egmont Foundation, The March of Dimes Birth Defects Foundation,

the Augustinus Foundation, and the Health Fund of the Danish Health Insurance Societies.

Fetal lung tissue We would like to acknowledge Professor DI Wilson, University of Southampton, for providing

fetal lung tissue.

Publically available data This study makes use of data generated by the GABRIEL Consortium.17

A full list of the investigators who contributed to the generation of the data is available from

http://www.cng.fr/gabriel/index.html. Funding for the project was provided by the European

Commission as part of GABRIEL (A multidisciplinary study to identify the genetic and

environmental causes of asthma in the European Community) contract number 018996

under the Integrated Program LSH-2004-1.2.5-1 Post genomic approaches to understand the

molecular basis of asthma aiming at a preventive or therapeutic control and the Wellcome

Trust under award 084703 (A second-generation genome-wide association study for

Asthma.

This study also makes use of data generated by the Wellcome Trust Case Control Consortium.

A full list of the investigators who contributed to the generation of the data is available

from www.wtccc.org.uk. Funding for the project was provided by the Wellcome Trust under

award 076113.


http://www.cng.fr/gabriel/index.html

http://www.wtccc.org.uk/

40

Funding

COPSAC COPSAC is funded by private and public research funds. The Lundbeck Foundation; the

Pharmacy Foundation of 1991; Augustinus Foundation; the Danish Medical Research Council

and The Danish Pediatric Asthma Centre provided the core support for the study.

The Danish National Birth Cohort (DNBC) control population DNBC was established with major funding from the Danish National Research Foundation.

Additional support for this cohort has been obtained from the Danish Pharmacists’ Fund, the

Egmont Foundation, the March of Dimes Birth Defects Foundation, the Augustinus Foundation

and the Health Fund of the Danish Health Insurance Societies.

MAAS MAAS was supported by the Asthma UK Grants No 301 (1995-1998), No 362 (1998-2001), No

01/012 (2001-2004), No 04/014 (2004-2007) and The Moulton Charitable Foundation

(2004-current); age 11 years clinical follow-up is funded by the Medical Research Council

(MRC) Grant G0601361.

Generation R The Generation R Study is conducted by the Erasmus Medical Center in close collaboration

with the School of Law and the Faculty of Social Sciences of Erasmus University Rotterdam,

the Municipal Health Service, Rotterdam area, the Rotterdam Homecare Foundation and the

Stichting Trombosedienst & Artsenlaboratorium Rijnmond (STAR-MDC; Rotterdam). The

generation and management of genotype data for the Generation R Study were performed at

the Genetic Laboratory of the Department of Internal Medicine at Erasmus Medical Center.

The Generation R Study is made possible by financial support from the Erasmus Medical

Center (Rotterdam), the Erasmus University Rotterdam, the Netherlands Organization for

Health Research and Development (ZonMw; 21000074), and the Lung Foundation

Netherlands (No 3.2.12.089; 2012).

Brooke Laboratory, Southampton The study was supported by the Medical Research Council (UK), Grant number G0900453.


41

IRB-BSC program on Computational Biology Josep M. Mercader was supported by Sara Borrell Fellowship from the Instituto Carlos III. This

work (DT, SB, JMM) has been supported by the grant SEV-2011-00067 of Severo Ochoa

Program, awarded by the Spanish Government.

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as a novel susceptibility gene for early childhood …...1 supplementary information a genome-wide...

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