UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA FACULTAD DE CIENCIAS DE LA SALUD TESIS DOCTORAL ANÁLISIS DE LOS POLIMORFISMOS DE LTC4S, ALOX5 Y ALOX5AP EN POBLACIÓN ASMÁTICA DE GRAN CANARIA JOSÉ ÁNGEL CUMPLIDO BONNY SERVICIO DE ALERGOLOGÍA HOSPITAL UNIVERSITARIO DE GRAN CANARIA DOCTOR NEGRÍN 2015
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UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA
FACULTAD DE CIENCIAS DE LA SALUD
TESIS DOCTORAL
ANÁLISIS DE LOS POLIMORFISMOS DE LTC4S, ALOX5 Y ALOX5AP EN POBLACIÓN ASMÁTICA DE GRAN
CANARIA
JOSÉ ÁNGEL CUMPLIDO BONNY
SERVICIO DE ALERGOLOGÍA HOSPITAL UNIVERSITARIO DE GRAN CANARIA
DOCTOR NEGRÍN
2015
A mi madre No elegimos la familia en la que nacemos y crecemos. Que buena suerte he tenido.
A mi mujer
Su alegría ante la vida es un estímulo constante.
AGRADECIMIENTOS
A mi Directora de tesis, Doctora Teresa Carrillo, por su inestimable ayuda,
apoyo y asesoramiento. Su sutil y educado apremio ha sido fundamental en la recta final.
Su constancia y capacidad de trabajo, un ejemplo a seguir.
A mi Director de tesis, Carlos Blanco, por su disponibilidad y ayuda constantes a
pesar de la distancia.
A María José Torres y su equipo en la unidad de investigación, gracias por el
minucioso trabajo realizado en la extracción y análisis del ADN.
A Estefanía Herrera y Luis Alberto Henríquez, por aclarar pacientemente mis
dudas sobre genética y estadística.
A mis compañeros del servicio de alergia: auxiliares, administrativos, médicos y
enfermeras. Todos son parte de esta Tesis, sin su colaboración no habría sido posible.
INDICE
Pág.
Lista de abreviaturas.......................................................................I
1. INTRODUCCION………………………………………………………….…...…1
1.1. Epidemiología……………….…………………………………………...2
1.2. Historia natural…….…………………………………………….……..6
1.3. Conceptos: Gravedad, control y exacerbaciones. ……..……6
1.3.1. Gravedad……………………………………………………………………….…..7
1.3.2. Control del asma..……………………………………………………….…….7
155. Sanak M, Pierzchalska M, Bazan-Socha S, Szczeklik A. Enhanced
expression of the leukotriene C(4) synthase due to overactive transcription
of an allelic variant associated with aspirin-intolerant asthma. Am J Respir
Cell Mol Biol. 2000;23(3):290-6.
156. Bauer J, Amelung P, Meyers D. Association of asthma susceptibility
and severity with a polymorphism in the leukotriene C4 (LTC4) synthase
gene. Am J Respir Crit Care Med 1999;159:A650–A.
Tesis Doctoral
José Ángel Cumplido Bonny Universidad de Las Palmas
116
157. Kim HB, Lee SY, Shim JY, Kim JH, Kang MJ, Hong SJ. The leukotriene
C4 synthase (A-444C) promoter polymorphism is associated with the
severity of exercise-induced asthma in Korean children. J Allergy Clin
Immunol. 2006;117(5):1191-2.
158. Isidoro-García M, Davila I, Moreno E, Laffond E, Lorente F, González-
Sarmiento R. Analysis of the leukotriene C4 synthase A-444C promoter
polymorphism in a Spanish population. J Allergy Clin Immunol. 2005;
115(1):206-7.
159. Zhang Y, Huang H, Huang J, Xiang Z, Yang M, Tian C, Fan H. The -
444A/C polymorphism in the LTC4S gene and the risk of asthma: a meta-
analysis. Arch Med Res. 2012;43(6):444-50.
FIG 1. ORs and their 95% CIs for the SNPs rs1624395 and rs1370128 in the
sequential addition of atopic asthma cases according to age at diagnosis.
J ALLERGY CLIN IMMUNOL
VOLUME 129, NUMBER 2
LETTERS TO THE EDITOR 573
IL-1 receptor–associated kinase 3 gene (IRAK3)variants associate with asthma in a replicationstudy in the Spanish population
To the Editor:Asthma is a chronic inflammatory disease associated with
genetic and environmental factors. Although the 12q13-24chromosome region had previously shown linkage to asthma,the IL-1 receptor–associated kinase 3 gene (IRAK3) has recentlyemerged as a susceptibility candidate for asthma as a result ofpositional cloning of persistent asthma with age of onset lessthan 13 years in Italian samples.1 IRAK3 encodes a proteinthat negatively regulates Toll-like receptor signaling pathwaysinvolved in innate host defense and in the control of adaptive im-mune responses. However, single nucleotide polymorphisms(SNPs) near IRAK3 have not reached the stringent significancethresholds in any of the genome-wide association studies(GWASs) performed in asthmatic patients.2 Here we aimed toreplicate the association of IRAK3 genetic variants with suscep-tibility to asthma in a case-control study with unrelated Spanishsubjects.For this purpose, DNA samples from 607 patients aged 5 years
or older with physician-diagnosed asthma and fulfilling theGlobal Initiative for Asthma guidelines (www.ginasthma.com)were collected from respiratorymedicine and allergy departmentsas part of the Genetics of Asthma (GOA) study in the Spanishpopulation. Samples from 1271 nonasthmatic patients without apersonal or familial history of allergic or pulmonary diseaseswere obtained from the Spanish National DNA Biobank (www.bancoadn.org), and these were used as control samples. Furthersample details can be found in the Methods section and TableE1 in this article’s Online Repository at www.jacionline.org.
Fifteen tagging SNPs (tSNPs) were selected and genotyped,as described elsewhere,3 to comprehensively survey commongene variation. All of them passed our quality control filtersand were considered for the analyses (see the Methods sectionand Table E2 in this article’s Online Repository at www.jacionline.org). Additionally, 83 European ancestry informativemarkers (EuroAIMs; see Table E3 in this article’s Online Re-pository at www.jacionline.org) were genotyped and used forprincipal component analysis with EIGENSOFT to derive thescores for the first principal component (PC1).4 Association ofindividual tSNPs was tested with the Cochran-Armitage trendtest, and then a logistic regression analysis was used to calculateodds ratios (ORs) with 95% CIs and to adjust for populationstratification by using the PC1 scores. Additionally, 15 untypedSNPs of the gene were imputed and tested for association. Hap-lotype associations were also tested to replicate previous find-ings.1 A false discovery rate (FDR) threshold of 5% wasestablished to limit the expected proportion of false-positive re-sults incurred in the study when a particular subject’s SNP testresult was considered significant. Further method details are de-scribed in the Methods section in this article’s OnlineRepository.For asthma, only 1 SNP (rs1168774) was nominally associated
but was not considered significant in the context of the multipletests performed (P 5 .038, FDR 5 42%, see Table E4 in this ar-ticle’s Online Repository at www.jacionline.org). However, 4tSNPs showed nominal association with atopic asthma (seeTable E4 for unadjusted results), 3 of them remaining significantafter adjusting for population stratification: rs1732877 (OR, 1.24;
95% CI, 1.04-1.46; P 5 .014), rs1624395 (OR, 1.22; 95% CI,1.03-1.45; P 5 .019), and rs1370128 (OR, 1.22; 95% CI, 1.03-1.44; P5 .019). Testing of untyped variants revealed 8 additionalSNPswith nominal association with atopic asthma (.014 <_P value<_ .048, see Table E4). All 11 SNPs were considered significantlyassociated with atopic asthma (FDR 5 2%) and showedmoderate-to-strong linkage disequilibrium (LD; 0.48 <_ r2 <_1.0). Three of them replicated previous findings (rs1624395,rs1370128, and rs2293657).1
A previous study suggested that association of IRAK3 withasthma varied with the age of onset.1 Analyzing the tSNPs repli-cating previous findings (rs1370128 and rs1624395), we ob-served that the effects were largest for 22 years of age atdiagnosis (Fig 1) both for rs1370128 (OR, 1.46; 95% CI, 1.14-1.87; P 5 .001; permuted P 5 .021) and rs1624395 (OR, 1.40;95% CI, 1.09-1.79; P 5 .007; permuted P 5 .043). Thereafter,considering only those atopic asthma cases diagnosed at 22 yearsof age or less, we observed nominal associations for 8 tSNPs, allshowing larger effects than when considering all atopic asthmacases (see Table E4). After adjusting for population stratification,all these associations remained significant: rs2701653 (OR, 1.51;95% CI, 1.04-2.19; P 5 .030), rs78503618 (OR, 1.57; 95% CI,1.15-2.16; P 5 .005), rs1732877 (OR, 1.43; 95% CI, 1.11-1.85; P 5 .007), rs1624395 (OR, 1.37; 95% CI, 1.07-1.76; P 5.013), rs1370128 (OR, 1.44; 95% CI, 1.12-1.84; P 5 .004),rs1152912 (OR, 1.36; 95% CI, 1.06-1.76; P 5 .018),rs1152913 (OR, 1.48; 95% CI, 1.14-1.91; P 5 .003), andrs1152916 (OR, 1.44; 95% CI, 1.11-1.86; P5 .007). On the con-trary, none of the tSNPs was significantly associated with atopicasthma diagnosed at age greater than 22 years (.228 <_ P value <_.971, not shown), suggesting that associations found with atopicasthma were likely to be due to the cases with an age at diagnosisof 22 years or less.Testing of untyped variants revealed 8 additional SNPs nom-
inally associated with atopic asthma diagnosed at age 22 years orless (.001 <_ P value <_ .040, Fig 2 and see Table E4). All 16 SNPsnominally associated with atopic asthma diagnosed at age 22years or less were considered significant (FDR <_ 2%), and mostof them were in moderate-to-strong LD. The exception werers2701653 and rs78503618 from the 59 region, which showed
FIG 2. Association P values (in log scale) of IRAK3 gene SNPs with atopic asthma diagnosed at age 22 years
or less and in a meta-analysis across Europeans. An LD plot of r2 values based on resequencing data is
shown for the 58 SNPs of IRAK3, as well as their relative position in the gene. Each diamond of the LD
plot represents the pairwise r2 correlation between SNPs.
J ALLERGY CLIN IMMUNOL
FEBRUARY 2012
574 LETTERS TO THE EDITOR
weak LD in pairwise comparisons between them (r25 0.001) andwith the rest of the SNPs of the gene (0.001 <_ r2 <_ 0.319).However, only the inclusion of the SNP rs2701653 as a covariatein logistic regression left a significant association for the 2 SNPsthat replicated previous findings (P 5 .012 and P 5 .007 forrs1624395 and rs1370128, respectively), indicating the presenceof independent associations.Haplotype testing of a previously associated 3-SNP region
(rs11465955, rs1624395 and rs1370128)1 replicated the associa-tion of the protective CGC haplotype (.001 <_P <_.032, see Table E5in this article’s Online Repository at www.jacionline.org).However, the association of the TAT risk haplotype1 was not repli-cated (P >_ .068), although it showed a significant effect in logisticregression (OR, 1.39; 95%CI, 1.07-1.81; P5 .013). Nevertheless,the CAT haplotype was detected as a risk haplotype for all asthmaphenotypes (P <_ .023, 1.37 <_ OR <_ .1.75; see Table E5). A furtherexploration suggested that the 3 SNPs included in haplotypes hadalmost perfect LD (D9) with 2 other nearby SNPs (rs2289134 andrs1732877), revealing similar effects when extending the haplo-types to intron 2 of the IRAK3 gene (see Table E5).
Because our results suggested that atopic asthma cases diag-nosed at age 22 years or less accounted for the discoveredassociations, we explored whether these associations had direc-tionally consistent effects with previous findings by performing ameta-analysis with data from candidate-gene case-control asso-ciation studies of unrelated patients with childhood and early-onset asthma (see the Methods section in this article’s OnlineRepository for further details).1,5 This analysis included 3384samples (736 cases and 2648 control subjects) and compared 7,3, and 5 SNPs overlapping between our samples and Sardinian,Italian, and Japanese subjects, respectively. This joint analysisonly evidenced allele and effect-wise replication among Euro-peans (Fig 2 and see Table E6 in this article’s Online Repositoryat www.jacionline.org), with 1 SNP associated with suggestivesignificance at a genome-wide level (rs1370128; OR, 1.54; 95%CI, 1.31-1.82; P 5 2.9 3 1027). Congruent with these results, astudy of 39 candidate genes in non-Hispanic white nuclear fami-lies with asthma6 reported a nominal association for 2 IRAK3SNPs: rs1821777, which shows an r2 value of 1 with rs2289134associated into haplotypes of 5 SNPs in our study, and
rs1152912, which is also associated in our study. However, alarge-scale asthma GWAS among Europeans reported a P valueof .07 for 1 IRAK3 SNP not tested in this study (rs17826057) ina meta-analysis combining cases with childhood-onset, later-on-set, severe, and occupational asthma, showing nonsignificant re-sults also when the analysis was restricted to asthma cases withonset at less than 16 years.7 Similarly, IRAK3 was not associatedwith childhood asthma among Japanese or Mexican nuclear fam-ilies in which most cases were atopic (90%).5,8 Although morestudies need to be accomplished, the nontransferability of theIRAK3 association with asthma between studies could be due todifferences in LD between populations, genetic and environmen-tal interactions, genetic heterogeneity, or the inadequate genecoverage of previous studies (see Table E7 in this article’s OnlineRepository at www.jacionline.org for gene coverage in Europeansin commercial arrays).8 Thus, despite the fact that SNPs from theIRAK3 gene are not among the GWAS hits for asthma to date,2 ourresults support the importance of this gene in patients with atopicasthma.In this study we performed the first independent replication of
IRAK3 association with asthma,1 examining extensively the mostcommon variation of the gene. Our results replicated the associa-tion of IRAK3 at the SNP and haplotype levels and evidenced thatother variants of the gene had independent effects in atopicasthma. These results were robust to population stratificationand to the differences in age and sex between cases and controlsubjects (not shown). Strikingly, the association of haplotypeswas not restricted to atopic asthma diagnosed at age 22 years orless but also was found among all asthma cases. However, theTAT risk haplotype was not replicated, which could be motivatedby our limited power to detect association on the basis of previousfindings1 (<60% for risks >_1.2 at a 2-sided P value of .05 and avariant frequency of 0.35 to 0.40) or the potential presence of pri-vate risk variants in Sardinians. Moreover, we could not distin-guish the causal variant because all but a few associated SNPswere in moderate-to-strong LD. Despite the fact that the testedtrait was not exactly the same as that analyzed among Italian sam-ples,1 because the persistency of asthma symptoms was not re-corded among GOA samples, the fact that we replicated thesame SNPs and the protective haplotype supports that these re-sults are unlikely to be spurious.In conclusion, our results confirm the importance of the IRAK3
gene in asthma pathogenesis in European populations, supportingthe hypothesis of shared risk factors among complex diseases3
and bolstering the relevance of the hygiene hypothesis in asthmadevelopment.9 Further research is needed to discern whether thisassociation is due to rare gene variants,10 to the biological func-tion of IRAK3, or to LDwith nearby genes, as well as its relevancein non-European populations.
We thank Carole Ober, Deborah Meyers, and the 4 reviewers who helped
improve the article and Tob�ıas Felipe for Fortran scripting.
Mar�ıa Pino-Yanes, MSca,b
Inmaculada S�anchez-Mach�ın, MDc
Jos�e Cumplido, MDd
Javier Figueroa, MDe
Mar�ıa Jos�e Torres-Galv�an, PhDf
Ruperto Gonz�alez, MDc
Almudena Corrales, CLTa,b
Orlando Acosta-Fern�andez, MDg
Jos�e Carlos Garc�ıa-Robaina, MDc
Teresa Carrillo, MDd
Anselmo S�anchez-Palacios, MDe
Jes�us Villar, MD, PhDa,h,i
Mariano Hern�andez, PhDj
Carlos Flores, PhDa,b
From aCIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid,
Spain; bthe Research Unit, Hospital Universitario NS de Candelaria, Tenerife, Spain;cthe Allergy Unit, Hospital Universitario NS de Candelaria, Tenerife, Spain; dthe Al-
lergy Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;ethe Allergy Unit, Hospital Universitario Insular de Gran Canaria, Spain; fthe Re-
search Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;gthe Neumology Unit, Hospital Universitario de Canarias, Tenerife, Spain; hthe Mul-
tidisciplinary Organ Dysfunction Evaluation Research Network (MODERN), Re-
search Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain;ithe Keenan Research Center, St Michael’s Hospital, Toronto, Ontario, Canada; andjthe Genetics Laboratory, Instituto Universitario de Enfermedades Tropicales y Salud
P�ublica de Canarias, Universidad de La Laguna, Tenerife, Spain. E-mail: cflores@ull.
es.
Supported by grants from the Ministry of Science and Innovation of Spain (PI081383)
and FUNCIS (27/07) and by a specific agreement between Instituto de Salud Carlos
III and Gobierno de Canarias (EMER07/001) under the ENCYT 2015 framework.
Disclosure of potential conflict of interest: The authors declare that they have no relevant
conflicts of interest.
REFERENCES
1. Balaci L, Spada MC, Olla N, Sole G, Loddo L, Anedda F, et al. IRAK-M is in-
volved in the pathogenesis of early-onset persistent asthma. Am J Hum Genet
2007;80:1103-14.
2. Ober C, Yao TC. The genetics of asthma and allergic disease: a 21st century per-
spective. Immunol Rev 2011;242:10-30.
3. Pino-Yanes M, Ma SF, Sun X, Tejera P, Corrales A, Blanco J, et al. Interleukin-
1 receptor-associated kinase 3 gene associates with susceptibility to acute lung in-
jury. Am J Respir Cell Mol Biol 2011;45:740-5.
4. Pino-Yanes M, Corrales A, Basald�ua S, Hern�andez A, Guerra L, Villar J, et al.
North African influences and potential bias in case-control association studies in
the Spanish population. PLoS One 2011;6:e18389.
5. Nakashima K, Hirota T, Obara K, Shimizu M, Jodo A, Kameda M, et al. An asso-
ciation study of asthma and related phenotypes with polymorphisms in negative
regulator molecules of the TLR signaling pathway. J Hum Genet 2006;51:284-91.
6. Rogers AJ, Raby BA, Lasky-Su JA, Murphy A, Lazarus R, Klanderman BJ, et al.
Assessing the reproducibility of asthma candidate gene associations, using
genome-wide data. Am J Respir Crit Care Med 2009;179:1084-90.
7. Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A
large-scale, consortium-based genomewide association study of asthma. N Engl
J Med 2010;363:1211-21.
8. Wu H, Romieu I, Shi M, Hancock DB, Li H, Sienra-Monge JJ, et al. Evaluation of
candidate genes in a genome-wide association study of childhood asthma in Mex-
Available online November 8, 2011.doi:10.1016/j.jaci.2011.10.001
The C11orf30-LRRC32 region is associatedwith total serum IgE levels in asthmaticpatients
To the Editor:Asthma is a chronic inflammatory respiratory disease charac-
terized by bronchial hyperresponsiveness, increased TH2 cyto-kine levels, and increased serum IgE levels. Atopic dermatitisor eczema is a chronic inflammatory skin disease characterizedby epidermal barrier dysfunction and IgE-mediated sensitization.The only published genome-wide association study (GWAS) ofatopic dermatitis identified rs7927894 on chromosome 11q13.5
§Only childhood cases from the Japanese study were considered in this analysis.
JALLERGYCLIN
IMMUNOL
FEBRUARY2012
575.e9
LETTERSTO
THEEDITOR
TABLE E7. Coverage of SNPs from the IRAK3 gene with MAFs of
5% or greater on commercial arrays used in asthma GWASs in
samples of European ancestry*
Coverage (%)
SNP array HapMap CEU Resequencing
Affymetrix 500K 63 29
Affymetrix Genome-Wide Human
SNP Array 6.0
93 71
Illumina HumanHap300� 77 19
Illumina HumanHap550� 83 35
Illumina HumanHap650Y 83 42
Illumina 1M-Duo 97 65
CEU, Ancestry from northern and western Europe.
*Includes studies described in Moffatt et al, 2007E20; Himes et al, 2009E21; Sleiman
et al, 2009E22; Li et al, 2010E23; Moffatt et al, 2010E24; Ferreira et al, 2011E25; and
Torgerson et al, 2011.E26
�Including the Illumina HumanCNV370-Duo as it was built on the Illumina
HumanHap300 BeadChip (317,000 SNPs) with an additional 52,000 markers aimed at
detecting copy number variations.
�Including the Illumina Human610 because it is similar to the Illumina
HumanHap550 with the addition of 60,000 markers to target regions with copy
number variations.
J ALLERGY CLIN IMMUNOL
VOLUME 129, NUMBER 2
LETTERS TO THE EDITOR 575.e10
BRIEF COMMUNICATION
No association between genetic ancestry and susceptibilityto asthma or atopy in Canary Islanders
María Pino-Yanes & Almudena Corrales &
José Cumplido & Ruperto González &
María José Torres-Galván &
Orlando Acosta Fernández &
Inmaculada Sánchez-Machín & Javier Figueroa &
Anselmo Sánchez-Palacios & Jesús Villar &
Mariano Hernández & Teresa Carrillo & Carlos Flores
Received: 12 April 2012 /Accepted: 6 June 2012 /Published online: 19 June 2012# Springer-Verlag 2012
Abstract Asthma is a complex respiratory disease charac-terized by chronic inflammation of airways and frequentlyassociated with atopic symptoms. The population from theCanary Islands, which has resulted from a recent admixtureof North African and Iberian populations, shows the highestprevalence of asthma and atopic symptoms among the Span-ish populations. Although environmental particularitieswould account for the majority of such disparity, geneticancestry might play a role in increasing the susceptibility of
asthma or atopy, as have been demonstrated in other recent-ly African-admixed populations. Here, we aimed to explorewhether genetic ancestry was associated with asthma orrelated traits in the Canary Islanders. For that, a total of734 DNA samples from unrelated individuals of the GOAstudy, self-reporting at least two generations of ancestorsfrom the Canary Islands (391 asthmatics and 343 controls),were successfully genotyped for 83 ancestry informativemarkers (AIMs), which allowed to precisely distinguishing
Electronic supplementary material The online version of this article(doi:10.1007/s00251-012-0631-3) contains supplementary material,which is available to authorized users.
M. Pino-Yanes :A. Corrales : J. Villar : C. FloresCIBER de Enfermedades Respiratorias,Instituto de Salud Carlos III,Madrid, Spain
M. Pino-Yanes :A. Corrales : C. Flores (*)Research Unit, Hospital Universitario N.S. de Candelaria,Carretera del Rosario s/n,38010 Santa Cruz, Tenerife, Spaine-mail: [email protected]
J. Cumplido : T. CarrilloAllergy Unit, Hospital Universitario Dr. Negrin,Las Palmas de Gran Canaria, Spain
R. González : I. Sánchez-MachínAllergy Unit, Hospital del Tórax,Complejo Hospitalario Universitario NS Candelaria,Tenerife, Spain
M. J. Torres-GalvánResearch Unit, Hospital Universitario Dr. Negrin,Las Palmas de Gran Canaria, Spain
O. A. FernándezNeumology Unit, Hospital Universitario de Canarias,Tenerife, Spain
J. Figueroa :A. Sánchez-PalaciosAllergy Unit, Hospital Universitario Insular de Gran Canaria,Las Palmas de Gran Canaria, Spain
J. VillarMultidisciplinary Organ Dysfunction Evaluation ResearchNetwork (MODERN), Research Unit,Hospital Universitario Dr. Negrin,Las Palmas de Gran Canaria, Spain
J. VillarKeenan Research Center, St. Michael’s Hospital,Toronto, ON, Canada
M. HernándezGenetics Laboratory, Instituto Universitario de EnfermedadesTropicales y Salud Pública de Canarias,Universidad de La Laguna,Tenerife, Spain
between North African and Iberian ancestries. No associa-tion was found between genetic ancestry and asthma orrelated traits after adjusting by demographic variables dif-fering among compared groups. Similarly, none of the indi-vidual AIMs was associated with asthma when results wereconsidered in the context of the multiple comparisons per-formed (0.005≤p value≤0.042; 0.221≤q value≤0.443). Ourresults suggest that if genetic ancestry were involved in thesusceptibility to asthma or related traits among CanaryIslanders, its effects would be modest. Larger studies, ex-amining more genetic variants, would be needed to exploresuch possibility.
Keywords Allergy . Genetic susceptibility . North Africa .
Admixture
Asthma is a complex respiratory disease characterized bychronic inflammation of airways and frequently associatedwith atopic symptoms (Ober 2005). Although environmen-tal factors are involved in its pathogenesis, familiar cluster-ing, twin studies, and genetic studies also support animportant genetic contribution to disease predisposition(Holberg et al. 1999; Ober and Hoffjan 2006; Bouzigon etal. 2010; Thomsen et al. 2010). The prevalence of asthmavaries widely around the world (1–18 %), and predictionsfor the coming years indicate that an increase is expected forWestern societies (Braman 2006; Pearce et al. 2007). InSpain, the prevalence of asthma symptoms has been esti-mated around 5–6 %, with considerable geographic varia-tion among regions (5–12 %) (The Spanish Group of theEuropean Asthma Study 1995). However, the highest prev-alence of asthma in adults from Spain has been found in theCanary Islands (17.2 %), based on the European Communi-ty Respiratory Health Survey (The Spanish Group of theEuropean Asthma Study 1995; Julia Serda et al. 2005). Inaddition, estimates of asthma prevalence in children aged 6–7 years are twofold higher in the Canary Islands (18.4 %) thanthe mean for the Spanish population (9.9 %) (Sanchez-Lermaet al. 2009). Similarly, the prevalence of atopy in the CanaryIslands is also elevated (40.6 %), compared to the mainlandSpanish populations (Julia-Serda et al. 2011).
The Canary Islands belong politically to Spain, in spite ofbeing located at about 1,000 km from the closest Europeanpoint in Iberian Peninsula and only 100 km off the North-west coast of Africa. These islands were inhabited by ab-original people related to the Berber populations at the timeof Spanish occupation in the fifteenth century, as pointed byhistorical, anthropological, archeological, and linguisticrecords (Navarro 1991). Genetic footprints of such North-west African influence (as a proxy for the aborigines) inCanary Islanders have been demonstrated in contemporaryinhabitants using classical (Flores et al. 2001) and autosomal
markers (Maca-Meyer et al. 2004; Fregel et al. 2005;Pino-Yanes et al. 2011), as well as with mitochondrial DNA(Rando et al. 1999) and the Y chromosome (Flores et al.2003). Recently, we have estimated North African admixturein Canary Islanders using almost a hundred ancestry informa-tive markers (AIMs), genetic loci showing large allele fre-quency differences between populations, selected fromdifferent regions of the autosomal genome (Pino-Yanes et al.2011). Intriguingly, our results suggested that North Africanancestry averaged 17±25 %, but showed a wide interindivid-ual variation ranging from 0 to 96%, which was interpreted asa trace of the violent way the islands were conquered andcolonized (Pino-Yanes et al. 2011).
Although the prevalence of asthma is generally low with-in countries from West Africa, individuals with West Afri-can ancestry have shown a higher prevalence of asthma andrelated traits when exposed to a Western style of livingcompared to European descent populations (NHLBI 2004).This has been explained as a consequence of gene–environ-ment interaction by which the admixed African populationwould carry risk alleles for asthma and related traits becausethese alleles would provide them an adaptation to a higherload of parasites in their original populations that are nolonger present in the new environment (Le Souef et al.2006). As a consequence, in a recent admixed population,it is expected that genomic segments originated from theancestral ethnic group with the higher risk will be enrichedwith risk alleles (Smith et al. 2004), which will be reflectedin a larger proportion of ancestry from the ancestral popu-lation in affected individuals compared to the unaffectedgroup. This hypothesis has been confirmed for AfricanAmerican and African Caribbean individuals, in which alarger African admixture has been associated with highersusceptibility for asthma and related traits and with lowerpulmonary function measures (Tsai et al. 2006; Vergara etal. 2009; Kumar et al. 2010, 2012; Flores et al. 2012).Similar to the West African populations, the prevalence ofasthma is generally low in the North African countries(NHLBI 2004), being estimated in 3–4 % for clinical diag-nosed asthma in Algeria, Morocco, and Tunisia (Nafti et al.2009). However, a trend for a markedly increase of preva-lence has been found in these populations over recent dec-ades due to the acquisition of a Western lifestyle andcontinued urban shifts (NHLBI 2004). In fact, for Morocco,the highest prevalence of asthma and asthma symptoms, aswell as of other allergic diseases, is found in Casablanca, thelargest city and the economic capital (Bouayad et al. 2006).This prevalence has been associated with an increase inurbanization and the acquisition of urban life style of living(Bouayad et al. 2006). In line with this data, the geneticexpression profile in peripheral blood leukocytes has beenfound to be profoundly changed by an urban living stylecompared to a rural living style in Northwest African
706 Immunogenetics (2012) 64:705–711
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populations (Idaghdour et al. 2008). Therefore, a higher riskfor asthma and related traits would be expected in popula-tions with North African genetic ancestry with Westernliving style.
Although the Canary Islands have particular climaticconditions that could promote the emergence of atopicsymptoms, it has been suggested that the genetic back-ground might also contribute to the high prevalence ofatopic symptoms and asthma in this population (Julia Serdaet al. 2005). Here, we aimed to analyze if genetic ancestry,assessed from autosomal AIMs, was associated with asthmaor related traits in a case–control sample of CanaryIslanders.
This study was approved by the ethics committees atparticipating hospitals, and written informed consent wasobtained from all subjects. This study was conducted usinga case–control design of 734 DNA samples from unrelatedindividuals reporting at least two generations of ancestorsborn in the Canary Islands. See Table 1 for the character-istics of subjects included in the study. Samples utilizedconstituted a subset of the Genetics of Asthma Study inthe Spanish population, described elsewhere (Pino-Yanes etal. 2012). Briefly, cases were represented by 391 physician-diagnosed asthmatic patients aged >5 years and fulfilling theGlobal Initiative for Asthma guidelines for diagnosis andclassification of asthma (http://www.ginasthma.com). Thesesamples were collected from respiratory medicine and allergydepartments from hospitals belonging to the Spanish NationalHealth System. Only individuals, who reported having at leasttwo generations of ancestors born in the Canary Islands, wereincluded in this study. Demographical and clinical data
recorded included: age, sex, smoking habits, age at diagnosisof the disease, rhinitis, atopic dermatitis, basal forced expira-tory volume in 1 s (FEV1), serum total IgE levels, and thesymptomatic treatment. Atopy was confirmed by a positiveskin prick test (SPT, a wheal with a diameter 3 mmgreater than the saline control) and/or elevated specificserum IgE levels (>0.35 UI mL−1) to at least one ofseven common allergens. Particularly, allergens evaluat-ed for specific IgE were: dust mite (Dermatophagoidespteronyssinus, Dermatophagoides farinae, Glycyphagusspp, Blomia tropicalis, Acarus siro, Tyrophagus putres-centiae, Lepidoglyphus destructor, and Euroglyphusmaynei), epithelium (Felis domesticus, Canis familiaris,Equus caballus, and Oryctolagus cuniculus), pollen(Olea europaea, Salsola spp, Lolium perenne, Artemisiavulgaris, Parietaria spp, Platanus spp, Chenopodiumspp, Plantago spp, Rumex spp, and Cupressus spp),fungi (Penicillium notatum, Alternaria alternate, Asper-gillus fumigatus, and Cladosporium herbarum), food(Cow's milk, hen's egg, peanut, soybean, wheat, fish,shrimp, crabs, lobster, clams, oysters, mussels, banana, chest-nut, and kiwi), latex and cockroaches (Blattella germanica).
Controls consisted of 343 DNA samples from nonasth-matic unrelated adults, which were obtained from blood bankdonors who did not report a personal or familiar medicalhistory for allergic or pulmonary diseases and self-reportingat least two generations of ancestors born in the CanaryIslands. A total of 93 European AIMs (from now on referredas EuroAIMs), allowing to distinguish between North Africanand Iberian ancestries, were genotyped in cases and controlsusing iPLEX™ Gold assays on MassARRAY system by the
Table 1 Relevant demographicand clinical features of samples
In parentheses, total number ofsubjects with available data
NA not available, P25 percentile25, P75 percentile 75aχ2 testbMann–Whitney U test
Spanish National Genotyping Center, Santiago de Compos-tela Node (CeGen, http://www.cegen.org/) (Pino-Yanes et al.2011). Briefly, iPLEX™ assays were scanned by MALDI-TOF mass spectrometry, and individual SNP genotype callswere automatically generated using Sequenom Typer 3.4™software. Samples from the Coriell Institute for Medical Re-search (http://www.coriell.org) were included to test allelecalling reliability of this platform.
Departures from Hardy–Weinberg equilibrium (HWE)were evaluated separately in cases and controls using anexact test (Wigginton et al. 2005), by means of the SNPIn-fostats software (Pino-Yanes et al. 2011). Given its higherdiscriminatory power to detect differences among closelyrelated populations, such as North Africans and Iberians(Maca-Meyer et al. 2004), than alternative algorithms basedon the assignation of individuals to discrete populations(Heath et al. 2008; Li and Yu 2008; Price et al. 2008),principal component analysis (PCA) was utilized to deriveindividual ancestry estimates as scores of the first principalcomponent as the major axis covering the Iberian-NorthAfrican genetic differences (Pino-Yanes et al. 2011). Forthis purpose, data for the same set of EuroAIMs fromindividuals, self-declaring two generations of ancestorsfrom the Iberian Peninsula (n0889) and Morocco in NorthAfrica (n068), were jointly analyzed with the case–controlsample to serve as reference populations (Pino-Yanes et al.2011). EIGENSOFT was used to assess PCA and to test theassociation of ancestry estimates with asthma and atopic traitsbetween cases and controls by means of ANOVA statistics(Price et al. 2006). Then, in order to adjust this association,clinical and demographic variables were included as covari-ates along with ancestry estimates in regression models utiliz-ing SPSS 15.0 (SPSS Inc., Chicago, IL). Given that geneticancestry estimates represented a mean value from the genome,we also explored the possibility of having local differences ingenetic ancestry that could be detected by using the individualEuroAIMs as proxies. For that, SNPassoc (Gonzalez et al.2007) was employed to test the association of individualEuroAIMs with asthma, adjusting for the genetic ancestryestimates by means of logistic regression models. Finally, inorder to limit the expected proportion of false positives in-curred in the study when a particular individual EuroAIMassociation test was called significant and to account for themultiple phenotypes tested for association with genetic ances-try, a false discovery rate was computed using QVALUE(Storey and Tibshirani 2003), and a threshold q value of0.05 was established to declare significance.
Eight SNPs gave poor-quality genotype data duringgenotyping (rs1032143, rs1073321, rs12502036,rs1364394, rs153595, rs1854226, rs2171209, andrs7108371) and were discarded from the study. In addition,two SNPs that deviated significantly from HWE, after con-sidering a multiple correction threshold of p<6x10-4 (0.05/85)
at least in one study sample (rs7277342 and rs2596501), werealso dropped from the analyses. Therefore, further analysesincluded the remaining 83 EuroAIMs, which had an averagecompletion rate of 98.0 % (P25–P75097.5–98.6 %), for a totalof 734 samples (mean sample completion rate098.0 %, P25–P75098.8–100.0%). As previous findings have showed a highcorrelation between individual ancestry estimates obtainedwith the full set of 93 EuroAIMs and with a reduced subsetof as few as 65 markers (Pino-Yanes et al. 2011), the finalnumber of EuroAIMs considered was still adequate for thepurpose of the study. See Table S1 in the Online Resource forfurther details.
The comparison of genetic ancestries between asthmaticor atopic asthma patients with the control group did notreveal significant differences. Similarly, no association wasfound between genetic ancestry and a positive result neitherfor atopy, SPT or rhinitis (Table 2), nor with serum levels oftotal IgE or with pulmonary function, measured by thepercentage of predicted FEV1 (Table 2). Genetic ancestrywas associated at nominal significance with a positive resultfor specific IgE among asthmatics (p00.004) (Table 2).Although this comparison was still significant after consider-ing all traits tested (q00.032), this result was considerednonsignificant after adjusting for age as a covariate (p00.056). We further explored the effect of age by subdividingthe sample according to age quartiles (Q): ≤23 years (Q1), 24–32 years (Q2), 33–44 years (Q3), and >44 years (Q4). Theresults were not significant in any of the age groups, and thedirectionality of effects was different between subsamples,particularly between the group of individuals with ≤23 yearsversus all the rest of analyzed groups (≥24 years) (Table S2 inthe Online Resource). In fact, genetic ancestry was signifi-cantly associated with a positive result for specific IgE amongasthmatics with ≥24 years (p value00.008 and beta0−29.82).This result may point to a modifier effect of age in the associ-ation of genetic ancestry with a positive result for specific IgEamong asthmatics. While several evidences suggest that demo-graphic and environmental factors influence total and specificIgE, and that specific IgE levels decrease with the increasing ofage (Omenaas et al. 1994; Borish et al. 2005), due to the limitedpower to detect association after sample was subdivided(<22 % on each subsample), future studies with larger samplesizes would be needed to determine if such effect is genuine.
Despite no evidence of association was found betweengenetic ancestry and asthma risk among Canary Islanders,we then determined whether any of the individual Euro-AIMs was associated with asthma. Adjusting the associationof each marker by ancestry differences between patients andcontrols, seven EuroAIMs were nominally associated(0.005≤p value≤0.042), although none of these associationswas considered significant in the context of the multiplecomparisons performed (0.221≤q value≤0.443) (Table S3in the Online Resource).
In this study, we have explored for the first time whethergenetic ancestry in Canary Islanders was associated withasthma and related traits. Despite the high prevalence ofasthmatic and atopic symptoms estimated for this popula-tion compared to the rest of populations from Spain (JuliaSerda et al. 2005), no association was found between genet-ic ancestry and asthma or related traits in Canary Islanders.Similarly, none of the individual EuroAIMs showed associ-ations with asthma. These results are in agreement withprevious observations based on self-reported informationfrom grandparental place of birth, where subjects with twogenerations of ancestors born in the Canary Islands did notshow increased asthma risk when compared with subjectswith ancestors born elsewhere (Julia Serda et al. 2005).
Although the Canary Islands have particular climaticconditions, such as high humidity and stable warm temper-atures that provide ideal conditions for mites and molds,which favor the emergence of atopic and asthmatic symp-toms in the Canary Islands (Sanchez-Lerma et al. 2009),genetic factors are also be involved in this disease. Accord-ing to the hygiene hypothesis (Strachan 1989), we speculat-ed that genetic African ancestry in the Canary Islanderscould provide a higher risk for asthma and atopy becausethe African ancestral population would have adapted to apathogen-rich environment that no longer exists in the cur-rent urban society in the Canary Islands. In line with this, arecent study has shown that the pathogenic environment(mostly the exposure to helminthes) is the predominant driverof local adaptation, even stronger than climate (Fumagalli etal. 2011). In addition, allele frequencies in genes involved inimmunity response have been strongly correlated with patho-genic environment (Fumagalli et al. 2011). Remarkably, it hasbeen revealed that several genes associated at the genome-wide level with helminth diversity were also associated withsusceptibility for asthma and atopy (Fumagalli et al. 2010).Under this scenario, the helminth-driven selective pressure isexpected to favor individuals carrying alleles that allow a
strong Th2 response and, therefore, to promote the transmis-sion and spread of asthma-susceptibility variants (Fumagalli etal. 2010).
Previous studies have examined the association of genet-ic ancestry and asthma susceptibility in recently admixedpopulations, observing a difference of 5–6 % in Africanancestry between cases and controls in independent Africandescent populations from the USA and the Caribbean(Vergara et al. 2009; Flores et al. 2012). Our statistical powerwas adequate (≥80 %) to detect differences in North Africanadmixture between cases and controls with effects even lowerthan the previously observed (up to 3.2 % of differences inNorth African admixture, i.e., a standardized effect of ≥0.2)(Fig. 1). However, in the hypothetical case that the geneticancestry had a true association with susceptibility to asthma orrelated traits, the presence of smaller effects cannot be dis-carded. Similarly, it is likely that the use of a larger number ofmarkers will allow estimating further ancestries among
Table 2 Association betweengenetic ancestry and asthmaticand atopic phenotypes
aLogistic regression model in-cluding gender and smokinghabits as covariatesbLogistic regression model in-cluding the age as a covariatecLinear regression model includ-ing the age as a covariate
Clinical variable Association p value (q value) Beta
Specific IgE (positiveness) 0.004 (0.032) 0.056b (0.448) −18.66
Total IgE levels (log scale) 0.524 (0.599) 0.958c (0.958) −0.13
FEV1 (%) 0.609 (0.609) 0.576c (0.889) 43.95
Fig. 1 Statistical power to detect differences in the genetic ancestryproportions by standardized effects for the sample size utilized (391cases and 343 controls)
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Canary Islanders. In this study, samples from different loca-tions in Morocco were used as a proxy for the aboriginalpopulation inhabiting the Canary Islands before the conquest.Although this is a reasonable assumption given the evidencefrom the historical records (Chejne 1974; Flores et al. 2001)and the numerous previous genetic studies (Rando et al. 1999;Flores et al. 2001, 2003, 2004; Alonso et al. 2005; Adams etal. 2008; Fregel et al. 2009), this obviously constitutes asimplification of the heterogeneous African influences thathave affected the Canary Islands populations. Therefore, it islikely that further sampling of North African regions and thetyping of additional markers might allow identifying othergenetic influences in Canary Islanders, which could clarify ifa true relationship between genetic ancestry and asthma existsin this population. In addition, another limitation of this studyis that the socioeconomic status of individuals was notrecorded, and therefore, its effects on asthma risk were notexplored. Given that the Spanish National Health Systemguarantees free healthcare access to the whole population,not only for treatment and rehabilitation but also for thepromotion of health and prevention of disease, a limited effecton asthma risk is expected.
In conclusion, no association was found between geneticancestry and asthma or related traits among Canary Islanders.Larger studies examining more genetic variants would beneeded to explore whether genetic ancestry has modest effectsin increasing asthma risk in the Canary Islanders.
Acknowledgments We thank David Comas and Jose M. Larruga forproviding Northwest African samples. This work was supported bygrants from the Health Institute “Carlos III” (FIS PI08/1383, FIS PI11/00623) and cofinanced by the European Regional Development Funds,“Away of making Europe” from the European Union, by a grant fromFundación Canaria de Investigación y Salud (FUNCIS 27/07), and by aspecific agreement between Instituto de Salud Carlos III and Gobiernode Canarias (EMER07/001).
Conflict of interest None.
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Assessing the Validity of Asthma Associations for EightCandidate Genes and Age at Diagnosis EffectsMarıa Pino-Yanes1,2¤, Almudena Corrales1,2, Jose Cumplido3, Paloma Poza4,
Inmaculada Sanchez-Machın4, Anselmo Sanchez-Palacios5, Javier Figueroa5,
Orlando Acosta-Fernandez6, Nisa Buset7, Jose Carlos Garcıa-Robaina8, Mariano Hernandez9,
Jesus Villar1,7,10, Teresa Carrillo3, Carlos Flores1,2,9*
1 Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain, 2 Research Unit, Hospital Universitario
Nuestra Senora de Candelaria, Santa Cruz de Tenerife, Spain, 3 Allergy Unit, Hospital Universitario Dr. Negrın, Las Palmas de Gran Canaria, Spain, 4 Allergy Management
Unit, Hospital del Torax (Ofra), Santa Cruz de Tenerife, Spain, 5 Allergy Unit, Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria, Spain, 6 Neumology
Unit, Hospital Universitario de Canarias, San Cristobal de La Laguna, Spain, 7 Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain, 8 Allergy
Unit, Hospital Universitario Nuestra Senora de Candelaria, Santa Cruz de Tenerife, Spain, 9 Applied Genomics Group (G2A), Genetics Laboratory, Instituto Universitario de
Enfermedades Tropicales y Salud Publica de Canarias, Universidad de La Laguna, San Cristobal de La Laguna, Spain, 10 Keenan Research Center, St. Michael’ Hospital,
Toronto, Ontario, Canada
Abstract
Background: Before the advent of genome-wide association studies (GWAS), ADAM33, ADRB2, CD14, MS4A2 (alias FCER1B),IL13, IL4, IL4R, and TNF constituted the most replicated non-HLA candidate genes with asthma and related traits. However,except for the IL13-IL4 region, none of these genes have been found in close proximity of genome-wide significant hitsamong GWAS for asthma or related traits. Here we aimed to assess the reproducibility of these asthma associations and totest if associations were more evident considering the effect of age at diagnosis.
Methodology/Principal Findings: We systematically evaluated 286 common single nucleotide polymorphisms (SNPs) ofthese 8 genes in a sample of 1,865 unrelated Spanish individuals (606 asthmatics and 1,259 controls). We found that variantsat MS4A2, IL4R and ADAM33 genes demonstrated varying association effects with the age at diagnosis of asthma, with 10SNPs showing study-wise significance after the multiple comparison adjustment. In addition, in silico replication with GWASdata supported the association of IL4R.
Conclusions/Significance: Our results support the important role of MS4A2, IL4R and ADAM33 genes in asthma and/oratopy susceptibility. However, additional studies in larger samples sets are needed to firmly implicate these genes in asthmasusceptibility, and also to identify the causal variation underlying the associations found.
Citation: Pino-Yanes M, Corrales A, Cumplido J, Poza P, Sanchez-Machın I, et al. (2013) Assessing the Validity of Asthma Associations for Eight Candidate Genesand Age at Diagnosis Effects. PLoS ONE 8(9): e73157. doi:10.1371/journal.pone.0073157
Editor: Gualtiero I. Colombo, Centro Cardiologico Monzino IRCCS, Italy
Received February 13, 2013; Accepted July 18, 2013; Published September 9, 2013
Copyright: � 2013 Pino-Yanes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funded by grants from the Carlos III Health Institute (http://www.isciii.es) and cofounded by the European Regional Development Fund (FEDER) "A wayof making Europe": PI081383 and EMER07/001, and by a grant from Fundacion Canaria de Investigacion y Salud FUNCIS 27/07 (http://www.funcis.org) to CF. MPYwas funded by a postdoctoral fellowship from Fundacion Ramon Areces (http://www.fundacionareces.es). The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
aThe National Heart Lung and Blood Institute’s (NHLBI) Programs for Genomic Applications (http://pga.gs.washington.edu).bThe Inate Immunity NHLBI Program for PGA (https://regepi.bwh.harvard.edu/IIPGA2).cHapMap phase 2 (http://hapmap.ncbi.nlm.nih.gov) and data from 96 type 1 diabetes individuals [28].dThe NIEHS Environmental Genome Project (http://egp.gs.washington.edu).doi:10.1371/journal.pone.0073157.t001
Age at Diagnosis Effects in Asthma Candidate Genes
PLOS ONE | www.plosone.org 3 September 2013 | Volume 8 | Issue 9 | e73157
5 SNPs had been associated with asthma or related traits in
previous studies, but in this study we extended their association to
a Southwestern European population with noticeable North
African influences.
SA did not show any age at diagnosis cutoff that significantly
maximized the association of rs1800925 (in IL13–IL4) with atopic
asthma (lowest p-perm = 0.063). In contrast, SA revealed allele
effects peaking at the same age at diagnosis, 39 years (number of
cases = 427), for SNPs from MS4A2 and IL4R: rs569108 in
MS4A2 (p-perm = 0.005), and rs1805015 in IL4R (p-perm = 0.001).
However, SA revealed allele effects peaking at a different age at
diagnosis for the SNPs from the other two genes: rs2071590 in
LTA-TNF showing a maximum at 26 years (p-perm = 0.002,
number of cases = 291), and rs2787095 in ADAM33 with a
maximum at 14 years (p-perm = 3.0E–04, number of cases =
155). The results obtained from the SA analyses using the quartiles
of the distribution of the age at diagnosis were equivalent to those
obtained using it as a continuous variable (data not shown).
Testing associations on the sub-sample of cases with the age at
diagnosis of asthma before the maxima determined by SA for each
(Table S3). Five of these SNPs (0.013# p-value #0.050) were
located in IL4R gene and only one of them constituted a positive
finding in previous studies. After conditioning these new associ-
ations from IL4R to the SNP rs1805015, only one SNP showed
independent association (rs3024676, p-value = 0.021). The
remaining 13 were all SNPs from ADAM33 (3.8E-5# p-value
#0.039), and 6 of them have been associated in at least one
previous study (Table S3 and Figure 1). After adjusting the
association of these 13 SNPs in ADAM33 that emerged with the
age at diagnosis cutoff for the SNP rs2787095, 7 SNPs (rs2787093,
rs628965, rs628977, rs630712, rs598418, rs2853209, and
rs603112) resulted independently associated from this SNP
(0.012# p-value #0.048). Therefore, the advantages of taking
into account the age at diagnosis varying effects for replication
studies in asthma were clearly evidenced in ADAM33, a gene for
which SNP-level replications are scarce in the literature [5,43].
Otherwise, we would have missed .50% of SNPs of this gene that
showed association in previous studies. For the LTA-TNF and
MS4A2 genes, we only observed subtle increases of effect sizes for
the SNPs that were revealed in our previous analyses, but did not
evidence more SNPs reaching nominal significance (Table S3 and
Figure 1). After a global FDR assessment accounting for all
comparisons performed, only 10 SNPs in MS4A2, IL4R and
ADAM33 genes showed an FDR ,5%, which were considered
associated at study-wise significance (Table 2). Among these, 7
SNPs were identified to be functional, as they were either
predicted to cause missense changes in the protein encoded or
had empirically demonstrated regulatory roles as deduced from
ENCODE project experimental data [42] (Table S4).
In order to provide evidence for replication at these loci, we
accessed the GABRIEL data, the largest GWAS meta-analysis in
asthma performed in Europeans [17]. There, we were able to
allocate 11 out of the 51 SNPs that reached nominal significance
with asthma in our study (Table S5). Only the SNP rs1805012,
located in IL4R, demonstrated in silico replication in GABRIEL
(p = 5.7E-04), showing the same direction of effects as in our study.
Discussion
In this study, we have comprehensively analyzed the association
of 286 common variants of eight candidate genes with asthma and
atopic asthma in a case-control Spanish sample and found
associations for 10 SNPs in three of them (MS4A2, IL4R and
ADAM33) after considering all tests performed. We additionally
provided in silico replication for IL4R with GWAS data from the
GABRIEL study.
It is well known that the age-at-onset of asthma is associated
with different phenotypic characteristics [44], and it has recently
evidenced that age-varying genetic associations can cause non-
replication and, consequently, lead to missing important genetic
associations [45]. Therefore, here we re-evaluated the association
of these genes by restricting the analysis to case subjects with an
age at diagnosis of asthma before a cutoff that maximized allele
effects of replicated variants. This allowed us to verify that
association improved for certain genes, such as ADAM33, as
recently supported for other firm candidates [25,46,47], and also
to gain insight in the genetic complexity of asthma associations at
these candidate genes. Intriguingly, many of their effects peaked in
the range of age at diagnosis between 20 and 45 years,
coincidental with the age range with the maximum expression of
the disease [48,49]. It remains to be solved whether or not true
biological mechanisms underlie this and previous observations
[17,25,46]. Nevertheless, our results suggest that it will be worth
considering the disease age at diagnosis in further studies, as well
as in the research of improved asthma treatment and prevention.
To identify firm susceptibility genes and understand the
biological processes underlying the development of the disease,
replication in independent well-powered studies is essential,
regardless of whether the first evidence of association was provided
by a GWAS study or a candidate gene survey [50]. Besides,
replication efforts allow testing the generalizability of findings in
other populations, and discovering novel genetic loci contributing
to phenotypic trait variability [51]. Particularly, testing the
associations in populations of recent African ancestry will likely
improve the detection of new risk variants [52], as they may offer
the opportunity to refine the signal or to allocate the causal
variants [53]. Our study aligns with these considerations, as it was
performed in a population with sizeable North African genetic
influences [28,54], and with a sample size representing a
substantially larger population of cases (.97%) than the vast
majority of prior published case-control studies of these genes in
unrelated individuals, although still far from optimal to detect
weak effects.
Under a simplistic scenario assuming complete LD of associated
SNPs with causal variants, the analyzed sample size provided a
70% power to detect a minimum risk of 1.45 for a risk allele
frequency of 45% with a two-sided p = 0.0012 significance level for
the primary outcome (asthma), and ranged from 14.6% to 52.6%
for the analyses in subset of cases with atopic asthma and asthma
before the age at diagnosis cutoff (Table S6). We acknowledge that
risk effects of this range are in the upper bound of those expected
for common variants in complex traits [55], which may have
contributed to our failure to detect associations for some of the
genes tested. Alternatively, our failure to find associations may
possibly be attributed to: i) Our impossibility to test their
association with more relevant traits or patient sub-samples (e.g.
asthma drug responses [56,57], environmental exposures [58]); ii)
The use of controls self-reporting no personal or familiar history of
pulmonary or allergic disease, but without a disease confirmation
based on a clinical characterization (e.g. lung function measure-
ments, SPT or specific IgE testing); iii) The lack of a true
association with asthma susceptibility, as has been suggested for
particularly relevant variants by meta-analyses [56]. Whichever is
correct, a recently published study with on a similar sample size
showed positive and negative association results fully congruent
with ours [59]. In support of our results, we were able to replicate
in silico the association of a SNP in IL4R in the largest GWAS study
Age at Diagnosis Effects in Asthma Candidate Genes
PLOS ONE | www.plosone.org 4 September 2013 | Volume 8 | Issue 9 | e73157
published to date that included more than 25,000 Europeans [17].
This SNP from IL4R, as well as few others from the same gene that
were found associated in our study (rs1801275, rs1805015, and
rs3024676), also demonstrated congruent effects and significant
association in a recent GWAS of total IgE levels [60]. This
evidence supports that, despite the enormous efforts to disentangle
asthma genes such as those entailed by the GABRIEL study [17]
or the EVE consortium [19], many more asthma susceptibility
genes awaits its discovery.
Some recent replication studies focusing on candidate genes
have utilized available arrays for genome wide genotyping [61–63]
where common variants of many key asthma candidate genes
could be insufficiently covered. In this respect, Michel et al. [59]
indicated that only 37% of the previously associated SNPs from 14
candidate genes were captured by the array utilized by the same
authors on the first GWAS for asthma and, surprisingly, not a
single SNP from key asthma genes such as ADAM33, IL4 and
CD14 was contained in their array [8]. Only after extending the
study by further genotyping (and by imputation) on the same
samples of their GWAS, these authors were able to consistently
replicate many of the biological candidates that were missing from
their GWAS [59]. We confirmed that the coverage of published
GWAS for asthma performed in European populations to date has
been insufficient for ADAM33 (,30%), even in a best-case scenario
using the HapMap phase 2 data as a reference for comparisons
(Table S7). If array comparisons were made against the 1KGP
sequencing data [38], the coverage would be even lower
(Table S7). Besides, it is worth noting that the estimated coverage
of these genes might be inflated, as these were implicitly derived
for HapMap CEU data and the same data was used to inform the
SNP contents of the array, and we have assumed that the 100% of
SNPs contained in the array were successfully genotyped. Effects
similar to those related to the age-of-onset of asthma, exceptionally
explored [17], could have also contributed to find no association
for the genes explored here in the published GWAS for asthma.
In conclusion, here we found the association of 10 common
variants in three biological candidate genes (MS4A2, IL4R and
ADAM33) that attained study-wise significance, and one of them
was also supported by in silico replication in GWAS data.
Therefore, we provided independent support for their role as risk
factors for the amalgam of asthma phenotypes. Moreover, our
results evidenced the genetic complexity at some of these
susceptibility loci and the importance of considering age-at-onset
effects. Given the low statistical power of the present study,
Figure 1. P-values of association by chromosome position with A) asthma #14years for ADAM33, B) asthma #39 years for MS4A2and, C) asthma #39 years for IL4R. P-values are expressed in –log10 scale. The SNP number shown on the plot denotes the result for themost significant SNP for each gene and the results for the remaining were color coded to reflect their LD with this SNP based on pairwise r2 valuesfrom the 1KGP. Estimated recombination rates (from 1KGP) were also plotted on the right axis to reflect the local LD structure.doi:10.1371/journal.pone.0073157.g001
Table 2. Association summary of the 10 SNPs that resulted significantly associated with asthma after adjustments for the multiplecomparisons.
tSNPs are underlined.aAccording to NCBI build 36.3.bComputed for allele 1.cSNPs associated in previous studies.doi:10.1371/journal.pone.0073157.t002
Age at Diagnosis Effects in Asthma Candidate Genes
PLOS ONE | www.plosone.org 5 September 2013 | Volume 8 | Issue 9 | e73157
particularly limited in the case subset analyses when considering
the age at diagnosis, further studies will be needed to identify
causal variants and to unravel if these genes are truly associated
with asthma, with atopy or with both.
Supporting Information
Table S1 Relevant demographic and clinical features ofGOA samples.
(DOC)
Table S2 Information, completion rates and Hardy-Weinberg equilibrium (HWE) p-values for the tSNPs.(DOC)
Table S3 Association summary of SNPs with asthma,atopic asthma and asthma with age at diagnosis beforethe cutoff demonstrating the largest effects.(DOC)
Table S4 Functional annotation of the 10 associatedSNPs.
(DOC)
Table S5 In silico replication of the associated SNPscontained in the GABRIEL study.(DOC)
Table S6 Sample sizes and statistical power for eachanalysis performed in a subset of cases.
(DOC)
Table S7 Coverage of candidate genes on commercialarrays used in asthma GWAS in samples of Europeanancestry.
(DOC)
Text S1 Supplementary methods.
(DOC)
Acknowledgments
The authors want to thank Tobıas Felipe for programming, and Marıa
Torres (CeGen, Santiago de Compostela Node) for her exceptional
technical support with iPLEXH Gold assays. In addition, we would like to
express our gratitude to the Juvenile Diabetes Research Foundation/
Wellcome Trust Diabetes and Inflammation Laboratory (JDRF/WT DIL,
Cambridge Institute for Medical Research) for granting the access to the
MS4A2 re-sequencing data, and acknowledge that those who carried out
the original analysis and collection of the data bear no responsibility for the
analysis or interpretations of this study.
Author Contributions
Conceived and designed the experiments: MPY CF. Performed the
experiments: MPY AC JC PP ISM ASP JF OAF NB JCGR JV TC.
Analyzed the data: MPY CF. Contributed reagents/materials/analysis
tools: CF MH JV. Wrote the paper: MPY MH CF.
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Age at Diagnosis Effects in Asthma Candidate Genes
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Letter to the Editor
HLA-DRB1*15:01 allele protects from asthmasusceptibility
To the Editor:
Asthma is a chronic inflammatory disease associated withgenetic and environmental factors. The HLA locus is the mostpolymorphic and gene-dense region of the human genome andhas been associated with a large number of infectious andautoimmune diseases.1 Before the advent of genome-wideassociation studies (GWAS), HLA-DRB1 and HLA-DQB1 geneswere independently associated with asthma and related traits inseveral candidate gene association studies.2 The importance ofthese genes in the pathogenesis of asthma has recently beencorroborated by both individual and meta-analyzed GWAS.3-5
In spite of the evidence, the interpretation of these associationscan be problematic because of the complex relationship betweenthe allele at single-nucleotide polymorphisms (SNPs) and thevariation at classical HLA alleles. Interestingly, classic allelesassociatewith stronger effects than individual SNPs and constitutethe most likely functional variants.6 Here, we aimed to test theassociation of SNPs from HLA-DRB1 and HLA-DQB1 withasthma in Spanish samples, and to uncover the classic allelesthat are involved in the susceptibility to the disease.
In the discovery stage, DNA samples from 574 physician-diagnosed asthmatic patients from the Genetics of Asthma (GOA)study in the Spanish population were compared with samples of1186 nonasthmatic subjects obtained from the Spanish NationalDNA Biobank (www.bancoadn.org).7 An independent sample of568 asthma cases and 787 controls was used for replication. Forfurther description of the study design, see Fig E1, OnlineRepository text, and Table E1 in this article’s Online Repositoryat www.jacionline.org.
A total of 22 SNPs capable of predicting classic alleles fromHLA-DRB1 and HLA-DQB1 in European populations weregenotyped in the discovery sample using a combination ofdifferent methods (see Table E2 in this article’s Online Repositoryat www.jacionline.org).8 Their performance on predictingHLA-DRB1 and HLA-DQB1 classic alleles was first assessed bygenotyping 313 DNA samples from healthy Spanish individualswith paired data for classic alleles at a 4-digit resolution(Luminex, Austin, Tex). To impute classic alleles, we used areference data set with more than 2500 samples of Europeanancestry with dense SNP data and classical HLA allele typing.6
We used a new probabilistic approach (HLP*IMP:02), whichdelivers increased accuracy on European samples, even underconditions of reduced SNP coverage.6 The imputed alleles forthe training sample were compared with the observed classicalleles at 2-digit and 4-digit resolution. Only those classic allelesthat attained 80% or more sensitivity, specificity, and positive andnegative predictive values and that were common in the trainingsample (frequency >_5%) were tested for association.
The association of SNPs with asthma in the discovery samplewas tested using logistic regression models and includedpreviously obtained ancestry scores as covariates to adjust forpopulation stratification.7 In this stage, we performed multipletesting adjustments for SNPs and classic alleles by means of105 permutations in which case-control labels were swappedwhile maintaining the haplotype structure.
Seventeen SNPs were successfully genotyped and passedquality control checks (see Online Repository text andTable E2). Four of them were associated with asthma inthe discovery sample after multiple comparison adjustments(1.24 <_ odds ratio [OR] <_ 1.94, 2.8 3 1027 <_ P <_ .002)(see Table E3 and Fig E2 in this article’s Online Repository atwww.jacionline.org) and 2 of them were nominally significantin replication samples (P <_ .008) (Table I). A meta-analysisconfirmed the consistency of the effects for the association ofrs3135388 and rs6457617 with asthma (P 5 7.8 3 1025 and3.0 3 1025, respectively) (Table I), and conditionalregression analysis in the overall sample revealed theirindependent association (P 5 1.3 3 1024 for rs3135388 andP 5 1.5 3 1024 for rs6457617), consistent with their weaklinkage disequilibrium (r2 5 0.06).
In silico analysis of expression quantitative trait loci (eQTLs)revealed the role of associated SNPs as eQTLs for HLA-DRB1and/or HLA-DRB5 in lymphoblastoid cells derived fromEuropean individuals (see Table E4 in this article’s OnlineRepository at www.jacionline.org). The SNP rs3135388 wasalso located on an enhancer histone mark site in B-lymphocytecells and a transcription factor binding site as demonstrated byempirical data from the Encyclopedia of DNA Elements(ENCODE) (http://www.genome.gov). In addition, our resultsfor rs6457617 constitute a replication of a GWAS hit for asthmain Japanese.3 Interestingly, the 2 associated SNPs showedconsistent effects with those reported for lipid traits (see TableE5 in this article’s Online Repository at www.jacionline.org).
Quality assessment of the imputation of the 76 classic allelesdetected in the training sample (see Table E6 in this article’sOnline Repository at www.jacionline.org) revealed that 14 ofthe 25 common alleles passed our quality criteria and werefollowed-up for association analysis using logistic regressionmodels. Three classic alleles were associated with asthma in thediscovery sample after multiple testing adjustments (see TableE7 in this article’s Online Repository at www.jacionline.org):HLA-DQB1*06, HLA-DRB1*15, and HLA-DRB1*15:01(.001 <_ P <_ 1.0 3 1025). However, only 2 of them replicated atnominal significance: HLA-DRB1*15 and HLA-DRB1*15:01(P 5 .001 and .008, respectively). A meta-analysis confirmeda consistent protective effect of HLA-DRB1*15:01 forasthma across all the samples (OR, 0.66; 95% CI, 0.53-0.81;P5 6.73 1025). Interestingly,HLA-DRB1*15:01 has previouslybeen associated with an increased risk for multiple sclerosis andnarcolepsy. This result is consistent with other susceptibilitygenes that have been shown to have opposite effects in asthmaand autoimmune diseases (Table E5).9,10 However, this study isthe first revealing such an effect for a classic HLA allele.
Because a previous study has found an association betweenSNPs from the HLA region and sensitization to specificallergens,11 we performed a post hoc analysis for sensitizationto the 3 most common specific allergens within the asthmacases (see Online Repository text), and identified SNPrs2395175 as associated with the presence of sensitization todust mites, pollens, and animal epithelia in the discoverysample (P <_ .004), with both harmful and protective associations,depending on the allergen (see Table E8 in this article’sOnline Repository at www.jacionline.org). However, only the
*Number of individuals with positive and negative sensitization (sensitized: nonsensitized).
�Cochran’s Q test for heterogeneity.
�Significant P values in individual samples and those that revealed a consistent effect in the meta-analysis.
§Effect and interval derived from the RE model as suggested by the Q test.
kEffect and interval derived from the FE model as suggested by the Q test.
J ALLERGY CLIN IMMUNOL
nnn 2014
2 LETTER TO THE EDITOR
association with animal epithelia sensitization was replicated inindependent samples (P <_ .003), showing a large effectsize in the meta-analysis (OR, 2.00; 95% CI; 1.43-2.81; P 5 5.63 1025) (Table II). This SNP was also an eQTL for HLA-DRB1and/or HLA-DRB5 in lymphoblastoid cells (Table E4).
Our study has 2 main limitations. First, it had more than 80%statistical power for variants with an OR of more than 1.3, but notfor smaller effect sizes (see Fig E3 in this article’s OnlineRepository at www.jacionline.org). Second, association testingof the full spectrum of classic alleles at these 2 genes wasimpracticable, both because of sample size limitations andbecause of the imperfect predictive ability of the SNPs retainedfor classic allele imputation. Consequently, we analyzed only56% of common classic alleles in this population (see OnlineRepository text). It is possible that we missed other interestingassociations, as suggested by the fact that a weighted scoreincluding the 2 SNPs associated with asthma explained slightlyhigher phenotypic variance than did a model including the classicalleles (Nagelkerke’s R2 5 0.14 vs 0.10, respectively).
In summary, a deeper examination of HLA genes has revealed aclassic allele that shows pleiotropic effects for asthma and otherimmune-related diseases and likely constitutes a putative causalvariant. Analysis of SNPs also revealed shared genetic risk factorsbetween asthma and lipid levels. Finally, we provided evidencesupporting the role of HLA polymorphisms in specific allergicsensitization.
We thank Alexander Dilthey, Goncalo Abecasis, and Christian Fuchsberger
for their help with software tools, Servicio de Apoyo Inform�atico a la
Investigaci�on (ULL) for the HPC support, and Katherine K. Nishimura for
proofreading the manuscript.
Mar�ıa Pino-Yanes, PhDa,b,c
Almudena Corrales, CLTa,b
Marialbert Acosta-Herrera, MSca,b,d
Eva P�erez-Rodr�ıguez, MDe
Jos�e Cumplido, MDf
Paloma Campo, MD, PhDg
Amalia Barreto-Luis, BSa,b
Florentino S�anchez-Garc�ıa, MD, PhDh
Tob�ıas Felipe, PhDi
Inmaculada S�anchez-Mach�ın, MDj
In�es Quintela, MSck
Jos�e Carlos Garc�ıa-Robaina, MDe
Jes�us Villar, MD, PhDa,d
Miguel Blanca, MD, PhDg
Angel Carracedo, MD, PhDl,m
Teresa Carrillo, MDf
Carlos Flores, PhDa,b,n
From aCIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid,
Spain; bResearch Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain;cthe Department of Medicine, University of California, San Francisco, Calif; dthe
Research Unit, Multidisciplinary Organ Dysfunction Evaluation Research Network
(MODERN), Hospital Universitario Dr Negrin, Gran Canaria, Spain; ethe Allergy
Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain; fAllergy Unit,
Hospital Universitario Dr Negr�ın, Gran Canaria, Spain; gAllergy Service, Carlos
Haya Hospital, Malaga, Spain; hthe Immunology Unit, Hospital de Gran Canaria
Dr Negr�ın, Gran Canaria, Spain; iNorthWest Research Associates, Boulder, Colo;jthe Allergy Unit, Hospital del T�orax, Complejo Hospitalario Universitario NS
Candelaria, Tenerife, Spain; kGrupo de Medicina Xen�omica, CEGEN-ISCIII-
Universidade de Santiago de Compostela, Santiago de Compostela, Spain; lGrupo
de Medicina Xen�omica, Fundaci�on Galega de Medicina Xen�omica (SERGAS)-
CIBERER-Universidade de Santiago de Compostela, Santiago de Compostela, Spain;mCenter of Excellence in Genomic Medicine Research, King Abdulaziz University,
Jeddah, Saudi Arabia; and nApplied Genomics Group (G2A), Genetics Laboratory,
Instituto Universitario de Enfermedades Tropicales y Salud P�ublica de Canarias,
Universidad de La Laguna, Tenerife, Spain. E-mail: [email protected].
This work was supported by grants from the Health Institute ‘‘Carlos III’’ (grant no. FIS
PI08/1383 and grant no. FIS PI11/00623) and cofinanced by the European Regional
Development Funds, ‘‘Away of making Europe’’ from the European Union, and by a
specific agreement between Instituto de Salud Carlos III and Gobierno de Canarias
(grant no. EMER07/001). M. Pino-Yanes was supported by a postdoctoral fellowship
from Fundaci�on Ram�on Areces. M. Acosta-Herrera and A. Barreto-Luis were sup-
ported by fellowships from the Instituto de Salud Carlos III (grant no. FI11/00074
and grant no. FI12/00493, respectively).
Disclosure of potential conflict of interest: M. Pino-Yanes has received funding from the
Fundaci�on Ram�on Areces. A. Carracedo is employed at the University of Santiago,
which has received funding from the Ministry of Justice, the Ministry of Health,
and the European Union. The rest of the authors declare that they have no relevant
conflicts of interest.
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