ABSTRACT Several genome-wide association studies have been conducted to investigate the influence of genetic polymorphisms in the development of allergic diseases, but few of them have

included the X chromosome. The aim of present study was to perform an X chromosome-wide association study (X-WAS) for asthma symptoms. The study included 1307 children of which 294 were

asthma cases. DNA was genotyped using 2.5 HumanOmni Beadchip from Illumina. Statistical analyses were performed in PLINK 1.9, MACH 1.0 and Minimac2. The variant rs12007907 (g.29483892C>A)

in _IL1RAPL_ gene was suggestively associated with asthma symptoms in discovery set (odds ratio (OR)=0.49, 95% confidence interval (CI): 0.37–0.67; _P_=3.33 × 10−6). This result was

associated with asthma symptoms in discovery sample. In conclusion, the rs12007907 variant in _IL1RAPL_ gene was negatively associated with asthma and IL-13 production in our study and a

sex-specific association was observed in one of the validation samples. It suggests an effect on asthma susceptibility and may explain differences in severe asthma frequency between women

and men. SIMILAR CONTENT BEING VIEWED BY OTHERS EXOME VARIANTS ASSOCIATED WITH ASTHMA AND ALLERGY Article Open access 05 December 2022 TARGETED ANALYSIS OF GENOMIC REGIONS ENRICHED IN

AFRICAN ANCESTRY REVEALS NOVEL CLASSICAL HLA ALLELES ASSOCIATED WITH ASTHMA IN SOUTHWESTERN EUROPEANS Article Open access 08 December 2021 PRIORITIZATION OF CANDIDATE CAUSAL GENES FOR ASTHMA

IN SUSCEPTIBILITY LOCI DERIVED FROM UK BIOBANK Article Open access 08 June 2021 INTRODUCTION Asthma affects the low airways resulting in airflow obstruction, bronchial hyper-responsiveness

and inflammation.1 The most recent Global Asthma Report estimates that over 334 million people are affected by asthma worldwide.2 This prevalence is increasing in recent decades among

individuals living in industrialized countries and, more recently, in developing countries. Therefore, asthma is considered a public health problem.3, 4, 5, 6 Asthma is a complex disease and

the mechanisms responsible for its causation are not fully understood, although it is clear that genetic and environment factors play an important role in its pathogenesis.7, 8 Many

genome-wide association studies (GWAS) have been conducted to investigate the influence of genetic polymorphisms in the development of several diseases. It is estimated that almost 1400 GWAS

have identified ~2800 peak associations (_P_≤5.10−8) so far.9 However, <30% of these studies report the X chromosome in their analysis. This may happened for several reasons, including

the limited number of X chromosome single-nucleotide polymorphisms (SNPs) presented in some genotyping platforms and the difference in the number of X chromosome copies between men and

women, which complicate the association analysis. The X chromosome contains 2300 genes10 and almost 1,3M SNPs.4 Genes present in the X chromosome are associated with different genetic

diseases such as Duchenne muscular dystrophy,11 Hemophylia A12 and IPEX (immune dysfunction, polyendocrinopathy, enteropathy and X-linked) syndrome.13 In addition, this chromosome includes

genes that have been associated with allergic diseases, such as _FOXP3_14 and others, which may be of relevance in the context of asthma, such as the _TLR7_ and _TLR8_.15 Thus, analyzing

SNPs present in this chromosome may provide important insights regarding genetic variants associated with diseases and should not be neglected. This is of special interest in the asthma

context, where differences in prevalence, morbidity and severity of the disease according to gender have been reported.16, 17 These studies have shown that males are more affected during

early childhood but a gender shift has been observed at puberty, with a higher prevalence of asthma in females at adulthood. Factors contributing to such sex difference remain unclear and

may involve hormonal changes together with genetic predisposition. Considering the presence of important genes on the X chromosome, possible associations with complex diseases and

difficulties in analyzing X-linked markers in GWAS, several studies have analyzed the X chromosome separately.18, 19, 20 Therefore, the aim of this study was to perform an X chromosome-wide

association study (X-WAS) for asthma symptoms in childhood. To our knowledge, this is the first X-WAS for asthma conducted worldwide. METHODS STUDY DESIGN AND POPULATION CHARACTERIZATION As

described in previous publications,21, 22, 23 Social Changes, Asthma and Allergy in Latin America (SCAALA) is a research program conducted in Brazil and Ecuador. The Brazilian component

comprised a cohort of 1307 (705 males) unrelated children between 4 and 11 years old. Asthma symptoms were defined and classified as reported in a previous publication of SCAALA Salvador

studies.24 Briefly, children were classified as having current wheeze as previously described25, 26 by using phase II International Study of Asthma and Allergies in Childhood questionnaire

(wheezing in the last 12 months) and were considered to have current wheeze plus symptoms if parents reported wheezing in the previous 12 months and at least one of the following: (1)

diagnosis of asthma ever; (2) wheezing with exercise in the last 12 months; (3) four or more episodes of wheezing in the last 12 months; and (4) waking up at night because of wheezing in the

last 12 months. Ethical approval for this study was obtained from the Brazilian National Ethical Council, and written informed consent was obtained from the guardian of each child.

GENOTYPING AND QUALITY CONTROL DNA was extracted from peripheral blood using a commercial kit (Gentra Puregene Blood Kit (Qiagen, Germantown, ML, USA)). Subjects were genotyped using the

2.5HumanOmni Beadchip from Illumina (San Diego, CA, USA). There were 46 945 SNPs genotyped on X chromosome and 391 SNPs in pseudoautossomal regions between the X and Y chromosomes.

Genotyping quality control and data cleaning was performed as specified in Kehdy _et al._27 Closely related individuals (_n_=64) identified as in Kehdy _et al._27 were excluded. SNPs were

removed if MAF (minor allele frequency) ≤1% in males and females separately. SNPs were also excluded if MIF (missing frequency) was ≥2% threshold either the separated missing frequencies in

males and females (MIF males—MIF females). Genotyping data has been deposited at the European Genome-phenome Archive (EGA, http://www.ebi.ac.uk.ega/), which is hosted by the EBI, under

accession number EGAS00001001245. SEQUENCE ANNOTATIONS Comparative genomic data and regulatory features for the _IL1RAPL1_ gene (X: 29418224–29545784; GRCh37/hg19 reference sequence) were

obtained from both the Ensembl (http://www.ensembl.org) and University of California Santa Cruz (http://genome.ucsc.edu) genome browsers. SNP positions were cross-referenced with sequence

annotations, including genomic evolutionary rate profiling-constrained elements for 36 eutherian mammals (GERP-EPO low coverage),28 chromatin segmentation state, and enrichment for marks of

open chromatin (DNase I hypersensitive sites). These last two types of information were obtained from the ENCODE project.29 VALIDATION STUDIES Validation analyses were performed in

independent Brazil and US Latin American populations. The US Latin American samples dates were collected from Genes-environments and Admixture in Latino Americans study (GALA II). The GALA

II study is an ongoing multicenter case–control study of asthma in Latino children and adolescents (1893 asthma cases and 1881 controls), organized from the coordinating center based at the

University of California, San Francisco, CA, USA.30 Subjects were eligible if they were 8–21 years old and had no history of other lung or other chronic illnesses. Asthma was defined based

on physician diagnosis and report of symptoms and medication use within the past 2 years before the recruitment. Controls had no reported history of asthma, lung disease or chronic illness,

allergic disease (eczema, hives, hay fever and allergic rhinitis), no reported use of medication for allergies, and no reported symptoms of coughing, wheezing or shortness of breath in their

lifetime. The second replication set was the Pelotas birth cohort from Brazil.31 It is comprised of 1151 participants (343 asthma cases and 808 health individuals), which were interviewed

when they were 18 years of age. Asthma was defined based on the same criteria used to case definition in the SCAALA cohort. The third replication set was the Asthma and Allergic Rhinitis

Control Program in Bahia (ProAR). Cases were comprised of 420 patients with severe asthma and 305 patients with mild or intermittent control persistent asthma according to the Global

Initiative rating against Asthma32 from both genders and age ≥18 years, living in Salvador. Controls were comprised with 398 subjects without history of asthma, residing in Salvador and

unrelated of the cases. In this replication study, the rs12007907 genotyping was performed using TaqMan probe-based 5′-nuclease assays (Applied Biosystems, Foster City, CA, USA) in

QuantStudio 12k Flex equipment. CYTOKINE PRODUCTION Venous blood was collected into heparinised tubes and the collected cells were cultured as previously described by Figueiredo _et al._33

The production IL-5 and IL-13 upon pokeweed (PWM) stimulation was measured in whole-blood culture supernatants using commercially available antibody pairs and recombinant cytokine standards

(BD Biosciences Pharmingen, San Diego, CA, USA) by sandwich ELISA according to the manufacturer’s instructions. Cytokine concentrations were determined by interpolation of standard curves.33

STATISTICAL ANALYSIS Logistic regression analyses using additive genetic model for asthma were performed in PLINK 1.9 after filters application for quality control procedures. Analyses were

adjusted by sex, age and the first three principal components (PCs) delineated through Eigenstrat on 370 539 genome-wide autosomal SNPs as covariates. Because of sex-biased pattern of

admixture in Brazilian populations, with preferential mating between males with predominant European ancestry and women with predominant African or Amerindian ancestry,34 the non-European

ancestry inferred to the X chromosome tend to be higher than estimated for the autosomes.27 For this reason, the average local ancestry inferred for X chromosome has been used as covariate

in some analyzes. Details about the methodology used to infer X chromosome ancestry are described elsewhere.27 Based on the default of PLINK, the analysis were performed assuming no dosage

compensation. Thus male genotypes were coded as 0 or 1 and female genotypes were coded as 0, 1 or 2. Sex-stratified analyses were performed for taking into account the X-inactivation and

different effect sizes of X-linked SNPs between males and females. For this, data from each sex (cases and controls) were analyzed separately and then combined using a weighted Stouffer’s

method.35 To test for differences in effect size according to sex we performed tests of heterogeneity using the METAL program.35 The SNP imputation pre-phase for the first step was performed

in MACH 1.0 and the second phase in Minimac236, 37 and was run separately for males and females. For imputation we used as reference the African population (1000G.AFR, 20100804.tgz)

according 1000 genomes.4 We imputed with high confidence (R2=0.3). To obtain a normal distribution of cytokines values a log transformation was applied and the differences in cytokines

concentration among the rs12007907 genotypes were analyzed using linear regression adjusted for sex, age and the three PCs of genetic ancestry using 370 539 genome-wide autosomal SNPs.

GENE-BASED ASSOCIATION ANALYSIS Gene-based association tests were performed through VEGAS238 and MAGMA39 software. Briefly, this approach aggregate association signals considering all SNPs

within a gene while account for dependence between signals due to linkage disequilibrium (LD) to assign a level of significance for the association of the gene overall. In addition, MAGMA

also performs gene-based analysis using PC regression and this method was applied for comparison with the results based on _P_-value that were obtained in VEGAS2 and MAGMA. In all gene-based

analyzes empirical _P-_values were obtained through permutational methods, being performed 10.000 simulations in VEGAS2 and 100.000 in MAGMA. RESULTS The final data set, after quality

control for SNPs and individuals, included 35 410 SNPs and 1246 samples (280 cases). We have identified two markers in X chromosome suggestively associated with asthma symptoms (Figure 1).

Table 1 presents the top 20 SNPs ranked according to _P_-value. The strongest association was observed for the SNP rs12007907 (odds ratio (OR)=0.49, 95% confidence interval (CI): 0.37–0.67;

_P_=3.33 × 10−6), which is an intronic variant located in the _IL1RAPL1_ (IL-1 R acessory protein-like) gene. The second most significantly associated variant was the rs199522937 (OR=1.71,

95% CI: 1.35–2.14; _P_=5.11 × 10−6) located in an intergenic region 5 kb upstream of the _DACH2_ gene (Dachshund Family Transcription Factor). These results were virtually unchanged when

using the ancestry inferred to the X chromosome to adjust by population stratification (OR=0.49, 95% CI: 0.36–0.66; _P_=3.10 × 10−6 for rs12007907 and OR=1.71, 95% CI: 1.36–2.15; _P_=4.57 ×

10−6; for rs199522937). Likewise, the rs12007907 and rs199522937 SNPs remained as being most associated with asthma symptoms when the results obtained for males and females separately were

combined by the weighted Stouffer’s method (Supplementary Table 1). This supports validity of the findings when one takes into account the possibility of differential effect size and

direction between males and females as well X-inactivation (that is, males coding as 0 and 2 instead 0 and 1 as assumed in the analysis). The quantile–quantile-plot (QQ-plot) of the

_P_-values illustrates that the observed significant associations were beyond those expected by chance (Figure 2). Furthermore, estimated genomic inflation factor (_λ_) was=1, indicating

that population genetic structure had negligible impact on association results. Regional plot of logistic regression in genotyped and imputed data were performed for the most associated

region with asthma symptoms in X chromosome (Figure 3). Since the rs12007907 and other SNPs of _IL1RAPL1_ gene are very rare in non-African populations, we performed the imputation using the

African population (1000G.AFR, 20100804.tgz) according 1000 genomes as the reference population. The SNP rs12007907 is in high LD with other SNPs in the _IL1RAPL1_, suggesting that variants

of this gene are most likely associated with asthma symptoms in this genomic region. We carried out an _in silico_ analysis of the SNPs correlated (r2≥0.6) with rs12007907 to identify

putative functional variants in the _IL1RAPL1_ region. We evaluated various functional annotations in our sets of polymorphisms, including predicted chromatin state segmentation, predicted

DNAse hypersensitivity, and sequence conservation across mammals (Supplementary Table 2). This analysis revealed that rs714723 was the best candidate for a functional variant in the

rs12007907 LD block. This SNP is located within a CCCTC-binding transcription factor (CTCF) sequence and can alter CTCF affinity to this genomic region. Analysis using imputed data confirmed

the suggestive association of rs714723 and other variants in the rs12007907 LD block with symptoms of asthma in our population (Figure 3 and Supplementary Table 2). We followed-up the

association of rs12007907 with asthma in three independent validation cohorts for which information about this SNP were available. (Supplementary Table 3). In GALA II and Pelotas populations

the rs12007907 was not associated with asthma (OR=1.06, 95%CI: 0.92–1.23; _P_=0.420 and OR=1.26, 95% CI: 0.91–1.73; _P_=0.164, respectively). In the ProAr cohort, which was recruited in the

same city and is ethnically similar to the discovery sample of this study, although no significant association was observed between rs12007907 and asthma in general analysis (OR=0.96,

95%CI: 0.78–1.18; _P_=0.707), the rs12007907 was negatively associated with asthma among men (OR=0.45, 95% CI: 0.21–0.95; _P_=0.038) but not among women (OR=1.02, 95%CI: 0.82–1.26;

_P_=0.862). Otherwise, in the GALA II and Pelotas populations the results remained not significant when stratifying by sex. Also in the discovery set no differences according to sex were

observed for the association between rs12007907 and asthma (OR=0.59, 95%CI: 0.38–0.95; _P_=2,9 × 10−2 and OR=0.43, 95% CI: 0.29–0.64; _P_=2.4 × 10−5 among men and women, respectively). Since

Th2-type cytokine are associated with atopic asthma40 and considering that IL1 family members such as (IL33/ST2) components induce Th2 polarization,41 we analyzed the production of IL-5 and

IL-13 in PWM-stimulated whole-blood cultures according to the genotypes for our top SNP (rs12007907). As can be seen in Figure 4, the homozygote for A allele had a lower production of IL-13

in comparison with homozygote for C allele. The geometric mean of IL-13 concentration in AA genotype was 36% lower compared with the CC genotype (_P_=0.044). No statistically significant

differences were observed comparing the heterozygous (CA) to the CC genotype (_P_=0.278). The Kruskal–Wallis test also showed significant differences for IL-13 production between the

different genotypic groups of rs1200797 (_P_=0.044). No significant trend was found for IL-5 production according to rs12007907 genotype (data not shown). The results of heterogeneity tests

are shown in Supplementary Table 4. The rs56025647 was the top SNP for sex-differentiated effect size in risk for asthma on SCAALA cohort, although it did not reach a level of suggestive

association considering multiple heterogeneity tests (_P_=5.15 × 10−5). Among males, this SNP was positively associated with asthma symptoms (OR: 1.84, 95%CI: 1.25–2.71, _P_=1,88 × 10−3),

while among females it was negatively associated (OR: 0.56, 95%CI: 0.40–0.78, _P_=8,5 × 10−4). The regional plot of SNPs near to rs56025647 is showed in Supplementary Figure 1. The results

of gene-based analysis are showed in the Supplementary Figure 2 and Supplementary Table 5. The _NUDT10_ was the only significantly associated gene with asthma symptoms (empirical

_P-_value<0.01) in both analyses based on previously computed SNP _P_-values (MAGMA and VEGAS) as well as on PCs regression (MAGMA). The 100 SNPs most associated with asthma symptoms in

the discovery set and ranked according to the _P_-value are shown in Supplementary Table 6. DISCUSSION The X chromosome has been often overlooked in GWAS of asthma to date. An important

exception in this scenario is the Moffat _et al_ study,42 which performed X chromosome analysis in populations with predominant European ancestry but reported no statistically significant

association signals. Recently, a GWAS of asthma symptoms in Latin American children with remarkable African ancestry was carried out in the same cohort of present study, but the X chromosome

was excluded from the analyses.43 However, X-WAS studies can help clarify the role of several genes in the development of many diseases, including complex illnesses such as asthma. In this

context, this is to the best of our knowledge one of the first initiatives to perform an X-WAS for asthma symptoms in Latin American children to date. In our discovery sample two SNPs were

suggestively associated with asthma symptoms. The SNP rs199522937 are intergenic and located in an upstream region of _DACH2_ gene that encodes a 599 aminoacid protein that is a

transcriptional cofactor.42 _DACH2_ sequences suggest a role in abnormalities like cleft palate and megalocornea.43 While the association of this intergenic SNP with asthma seems unclear,

the top SNP in our study, located in the _IL1RAPL1_ gene region, suggest a possible causal effect on asthma susceptibility. The _IL1RAPL1_ (IL-1R accessory protein-like) is a gene that

encodes a protein with 696 aminoacids homologous to IL-1 receptor accessory proteins. _IL1RAPL_ was first identified in patients with X-linked mental retardation.44 Mutations in this gene

were also identified in patients with cognitive impairment. In these cases, a loss-of-function mutation in this gene impacts on an interleukin/MAP kinase cascade of modulatory

neurotransmitters affecting synapse structure leading to cognitive impairment.44 The exact function of _IL1RAPL1_ is not fully understood, but it is abundantly expressed in postnatal brain

structures and may interact with neuronal calcium sensor, thus possibly acting in regulation of exocytosis of secretory substances. The expression of IL1RAPL protein has been detected in the

brain, heart and muscle.45 Khan _et al_ showed that IL1RAPL can active c-Jun amino-terminal kinases (JNKs) but not extracellular signal-regulated kinases (ERKs) nor mitogen-activated

protein kinases (MAPKs).46 Although IL1R have high affinities for their ligands, the accessory proteins cannot do it directly. Furthermore, the function of IL1RAPL might be more related to

the Toll-like receptors subfamily than IL1R subfamily.45 Interestingly, _IL1RAPL1_ has already been reported in two previous GWAS to be associated with cardiovascular disease47 and autism.19

Although this gene belongs to the IL1R family, it has been poorly studied, possible due to its localization in the X chromosome and consequent exclusion from genome-wide analysis. The

rs12007907 is an intronic SNP which is in LD with others SNPs located in a regulatory region of the _IL1RAPL1_ gene, such as the rs714723 variant located within a CTCF sequence. Binding of

target sequence elements by CTCF can block the interaction between enhancers and promoters, therefore limiting the activity of enhancers to certain functional domains. CTCF can also act as a

chromatin barrier by preventing the spread of heterochromatin structures.48 The primary role of CTCF is thought to be in regulating the structure of chromatin. Therefore, SNPs located in

these regions could influence gene expression. However, further analyzes including functional assays are required to identify potential causal polymorphisms located in _IL1RAPL_ gene or even

in nearby genomic regions. Different IL-1 receptors, such as IL-1R, are found in the human genome and the IL1RAPL is a member of this subfamily. IL-1R is potential regulator of inflammation

and is critical for innate immunity and host defense against infections.31 IL-1R also play an important role in the regulation of TH2 cell in allergic airway inflammation.41 Previous GWASs

reported associations of _IL1R_ SNPs with asthma in different populations,49, 50, 51 suggesting that _IL1R_ gene may be involved in asthma susceptibility. Assuming that _IL1RAPL_ gene may

play a similar role of _IL1R_ in inflammatory responses mediated by TH2 cells, we decided to evaluate the association between the rs12007907 genotypes and the production of Th2-type

cytokines (IL-5 and IL-13) in our study population. Interestingly, the homozygous genotype for the rs1200797 allele negatively associated with asthma symptoms (AA genotype) was negatively

associated with IL-13 production. These results suggest a possible involvement of SNPs in _IL1RAPL1_ gene regulating IL-13 production, which needs to be further, investigated. However, that

could also explain the negative association observed for rs12007907 with asthma since IL-13 is a classical cytokine related to allergic asthma leading to bronchial hyper reactivity as well

as mucus production.52 In our validation samples we did not observe significant associations in general analysis. This can be explained, at least in part, by differential frequencies of

_IL1RAPL1_ SNPs between the populations evaluated. In fact, a pattern of biogeographical variation is observed for rs12007907, with allele frequencies varying from 37% among Africans, 3%

among Europeans and 7% among Amercan Indians. In SCAALA (the discovery sample) rs12007907 MAF (corresponding to the A allele) was 25%, reflecting the remarkable African contribution to this

admixed population.45 In the ProAr cohort the frequency of this SNP was 23%, which reflects the closest genetic similarity of this population with the discovery sample. However, in the GALA

II and Pelotas cohorts the MAFs were 7 and 11%, respectively, which may result in a lack of statistical power to detect an association. In addition, in the Pelotas cohort the outcome of

asthma symptoms was investigated during adolescence.31 Also, GALA II consists of adolescents and young adults,30 while the ProAr cohort is exclusively composed of adult individuals and

asthma phenotype different from SCAALA (SCAALA cohort which comprises mild asthmatics only while ProAr mild and severe asthmatics). Given the heterogeneity of asthma across different stages

of the life course, it is possible that genetic variants associated with childhood-onset disease are not the same implicated in late-onset disease, which is in accordance to evidence from

GWAS.42 Interestingly, in the analysis stratified by sex the rs12007907 was negatively associated with asthma among men in ProAR cohort, in agreement with that observed in the discovery set.

These results suggest that studies in admixed populations with predominantly non-European ancestry can shed light on genetic factors involved in gender differences for asthma prevalence.

Gene-based analysis revealed that _NUDT10_ was the most consistently associated with asthma symptoms in SCAALA-Salvador cohort. This gene belongs to NUDT gene family (NUDT='Nudix

[Nucleoside Diphosphate attached moiety ‘x’]-Type motif) and encode a phosphatase involved in the diphosphoinositol polyphosphates cell turnover.46 The diphosphoinositol polyphosphates have

been implicated in several cellular activities, such as homolog recombination, vesicle trafficking, apoptosis and response to environmental stress.47, 48, 49 Although association of the NUDT

genes with asthma has not been previously reported, the diphosphoinositol polyphosphates syntheses is activated in cells subjected to stress by hyperosmotic pressure.50 Such osmotic stress

may occur, for instance, in airways epithelial cells undergone to inadequate airway humidification caused by rapid breathing (for example, during exercise), breathing of dry/cold air and in

some airway diseases.51 Interestingly, the hyperosmolality of extracellular fluid is believed to play an inflammatory role in asthma and other diseases.52 However, further studies are needed

to clarify the role, if any, of _NUDT10_ gene in asthma risk. For complex diseases exhibiting sexual dimorphism, such as asthma, it is supposed that X-linked variants are more likely to

exhibit different effects between males and females.53 Therefore, we performed stratified analyzes in order to identify potential variants with different effect size in asthma symptoms

according to sex. The top SNP in the heterogeneity tests, rs56025647, is located in an intergenic region in Xq13. The role of genetic variants in this locus to asthma risk, however, is

unknown and needs to be further investigated. An important limitation of the present study is the relatively small sample size, which can lead to lack of power, especially because SNPs

generally have small effect size. Considering an OR of 1.5 and a significance level of 5.0 × 10−8, for example, the power reached by the study is only 30.5 and 41.2% for variants with

frequencies of 20 and 25%, respectively. Although not calculated, the power reached by the study in analyzes stratified by sex is even lower. Nevertheless, suggestive associations were

detected when correcting for multiple testing in overall sample. However, these results require further replication in more similar samples regarding their genetic background and age, which

shall be complemented with functional data to understand the impact of these herein described genes in asthma. CONCLUSION This is the first X chromosome-wide association study for asthma

carried out to date. Our findings showed that X-linked genes were suggestively associated with asthma symptoms in children, one of which belongs to IL1R’s family. This latter gene was also

associated with production of pro-inflammatory cytokine IL-13. Though the X chromosome is often excluded from GWAS, important differences in morbidity and severity of asthma has been

reported between males and females. This prevents the assessment of the impact of sex-linked genes in asthma. Thus our study may contribute to a better understanding of genetic factors

involved in asthma development and, in particular, to the observed gender differences. REFERENCES * Akdis CA : Allergy and hypersensitivity: mechanisms of allergic disease. _Curr Opin

Immunol_ 2006; 18: 718–726. Article  CAS  Google Scholar  * International Union Against Tuberculosis and Lung Disease and The International Study of Asthma and Allergies in Childhood: The

global asthma report 2014. Available from http://www.globalasthmareport.org (accessed March 2016). * Yazdanbakhsh M, Kremsner PG, van Ree R : Allergy, parasites, and the hygiene hypothesis.

_Science_ 2002; 296: 490–494. Article  CAS  Google Scholar  * Abecasis GR, Auton A, Brooks LD _et al_: An integrated map of genetic variation from 1,092 human genomes. _Nature_ 2012; 491:

56–65. Article  Google Scholar  * Pearce N, Ait-Khaled N, Beasley R _et al_: Worldwide trends in the prevalence of asthma symptoms: phase III of the International Study of Asthma and

Allergies in Childhood (ISAAC). _Thorax_ 2007; 62: 758–766. Article  Google Scholar  * Cooper PJ, Rodrigues LC, Cruz AA, Barreto ML : Asthma in Latin America: a public heath challenge and

research opportunity. _Allergy_ 2009; 64: 5–17. Article  CAS  Google Scholar  * Woolcock AJ, Peat JK : Evidence for the increase in asthma worldwide. _Ciba Found Symp_ 1997; 206: 122–134.

CAS  PubMed  Google Scholar  * Upton MN, McConnachie A, McSharry C _et al_: Intergenerational 20 year trends in the prevalence of asthma and hay fever in adults: the midspan family study

surveys of parents and offspring. _BMJ_ 2000; 321: 88–92. Article  CAS  Google Scholar  * Wise AL, Gyi L, Manolio TA : eXclusion: toward integrating the X chromosome in genome-wide

association analyses. _Am J Hum Genet_ 2013; 92: 643–647. Article  CAS  Google Scholar  * Ross MT, Grafham DV, Coffey AJ _et al_: The DNA sequence of the human X chromosome. _Nature_ 2005;

434: 325–337. Article  CAS  Google Scholar  * Bushby KM : Recent advances in understanding muscular dystrophy. _Arch Dis Child_ 1992; 67: 1310–1312. Article  CAS  Google Scholar  * Mannucci

PM, Tuddenham EG : The hemophilias—from royal genes to gene therapy. _N Engl J Med_ 2001; 344: 1773–1779. Article  CAS  Google Scholar  * Wildin RS, Freitas A : IPEX and FOXP3: clinical and

research perspectives. _J Autoimmun_ 2005; 25 (Suppl): 56–62. Article  CAS  Google Scholar  * Bottema RW, Kerkhof M, Reijmerink NE _et al_: Gene-gene interaction in regulatory T-cell

function in atopy and asthma development in childhood. _J Allergy Clin Immunol_ 2010; 126: 338–346. Article  CAS  Google Scholar  * Du X, Poltorak A, Wei Y, Beutler B : Three novel mammalian

toll-like receptors: gene structure, expression, and evolution. _Eur Cytokine Netw_ 2000; 11: 362–371. CAS  PubMed  Google Scholar  * Carey MA, Card JW, Voltz JW _et al_: It's all

about sex: gender, lung development and lung disease. _Trends Endocrinol Metab_ 2007; 18: 308–313. Article  CAS  Google Scholar  * Postma DS : Gender differences in asthma development and

progression. _Gend Med_ 2007; 4: S133–S146. Article  Google Scholar  * Chu X, Shen M, Xie F _et al_: An X chromosome-wide association analysis identifies variants in GPR174 as a risk factor

for Graves' disease. _J Med Genet_ 2013; 50: 479–485. Article  CAS  Google Scholar  * Chung RH, Ma D, Wang K _et al_: An X chromosome-wide association study in autism families

identifies TBL1X as a novel autism spectrum disorder candidate gene in males. _Mol Autism_ 2011; 2: 18. Article  CAS  Google Scholar  * Conde L, Foo JN, Riby J _et al_: X chromosome-wide

association study of follicular lymphoma. _Br J Haematol_ 2013; 162: 858–862. Article  Google Scholar  * Figueiredo CA, Alcantara-Neves NM, Veiga R _et al_: Spontaneous cytokine production

in children according to biological characteristics and environmental exposures. _Environ Health Perspect_ 2009; 117: 845–849. Article  CAS  Google Scholar  * Rodrigues LC, Newcombe PJ,

Cunha SS _et al_: Early infection with _Trichuris trichiura_ and allergen skin test reactivity in later childhood. _Clin Exp Allergy_ 2008; 38: 1769–1777. CAS  PubMed  Google Scholar  *

Barreto ML, Cunha SS, Alcantara-Neves N _et al_: Risk factors and immunological pathways for asthma and other allergic diseases in children: background and methodology of a longitudinal

study in a large urban center in Northeastern Brazil (Salvador-SCAALA study). _BMC Pulm Med_ 2006; 6: 15. Article  Google Scholar  * Figueiredo CA, Barreto ML, Rodrigues LC _et al_: Chronic

intestinal helminth infections are associated with immune hyporesponsiveness and induction of a regulatory network. _Infect Immun_ 2010; 78: 3160–3167. Article  CAS  Google Scholar  *

Alcantara-Neves NM, Veiga RV, Dattoli VC _et al_: The effect of single and multiple infections on atopy and wheezing in children. _J Allergy Clin Immunol_ 2012; 129: 359–367. Article  Google

Scholar  * Figueiredo CA, Amorim LD, Alcantara-Neves NM _et al_: Environmental conditions, immunologic phenotypes, atopy, and asthma: new evidence of how the hygiene hypothesis operates in

Latin America. _J Allergy Clin Immunol_ 2013; 131: 1064–1068. Article  Google Scholar  * Kehdy FS, Gouveia MH, Machado M _et al_: Origin and dynamics of admixture in Brazilians and its

effect on the pattern of deleterious mutations. _Proc Natl Acad Sci USA_ 2015; 112: 8696–8701. Article  CAS  Google Scholar  * Cooper GM, Stone EA, Asimenos G, Green ED, Batzoglou S, Sidow A

: Distribution and intensity of constraint in mammalian genomic sequence. _Genome Res_ 2005; 15: 901–913. Article  CAS  Google Scholar  * ENCODE Project Consortium: An integrated

encyclopedia of DNA elements in the human genome. _Nature_ 2012; 489: 57–74. Article  Google Scholar  * Nishimura KK, Galanter JM, Roth LA _et al_: Early-life air pollution and asthma risk

in minority children. The GALA II and SAGE II studies. _Am J Respir Crit Care Med_ 2013; 188: 309–318. Article  Google Scholar  * Victora CG, Barros FC : Cohort profile: the 1982 Pelotas

(Brazil) birth cohort study. _Int J Epidemiol_ 2006; 35: 237–242. Article  Google Scholar  * Van Ganse E, Antonicelli L, Zhang Q _et al_: Asthma-related resource use and cost by GINA

classification of severity in three European countries. _Respir Med_ 2006; 100: 140–147. Article  CAS  Google Scholar  * Figueiredo CA, Alcantara-Neves NM, Amorim LD _et al_: Evidence for a

modulatory effect of IL-10 on both Th1 and Th2 cytokine production: the role of the environment. _Clin Immunol_ 2011; 139: 57–64. Article  CAS  Google Scholar  * Salzano FM, Bortolini MC :

_The Evolution and Genetics of Latin American Populations_. New York: Cambridge Univ Press, 2002. Google Scholar  * Willer CJ, Li Y, Abecasis GR : METAL: fast and efficient meta-analysis of

genomewide association scans. _Bioinformatics_ 2010; 26: 2190–2191. Article  CAS  Google Scholar  * Fuchsberger C, Abecasis GR, Hinds DA : minimac2: faster genotype imputation.

_Bioinformatics_ 2014; 31: 782–784. Article  Google Scholar  * Howie B, Fuchsberger C, Stephens M, Marchini J, Abecasis GR : Fast and accurate genotype imputation in genome-wide association

studies through pre-phasing. _Nat Genet_ 2012; 44: 955–959. Article  CAS  Google Scholar  * Liu JZ, McRae AF, Nyholt DR _et al_: A versatile gene-based test for genome-wide association

studies. _Am J Hum Genet_ 2010; 87: 139–145. Article  CAS  Google Scholar  * de Leeuw CA, Mooij JM, Heskes T, Posthuma D : MAGMA: generalized gene-set analysis of GWAS data. _PLoS Comput

Biol_ 2015; 11: e1004219. Article  Google Scholar  * Barnes PJ : Th2 cytokines and asthma: an introduction. _Respir Res_ 2001; 2: 64–65. Article  CAS  Google Scholar  * Dunne A, O'Neill

LA : The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. _Sci STKE_ 2003; 2003: re3. PubMed  Google Scholar  * Moffatt MF,

Gut IG, Demenais F _et al_: A large-scale, consortium-based genomewide association study of asthma. _N Engl J Med_ 2010; 363: 1211–1221. Article  CAS  Google Scholar  * Costa GN, Dudbridge

F, Fiaccone RL _et al_: A genome-wide association study of asthma symptoms in Latin American children. _BMC Genet_ 2015; 16: 141. Article  Google Scholar  * Born TL, Smith DE, Garka KE,

Renshaw BR, Bertles JS, Sims JE : Identification and characterization of two members of a novel class of the interleukin-1 receptor (IL-1R) family. Delineation of a new class of

IL-1R-related proteins based on signaling. _J Biol Chem_ 2000; 275: 29946–29954. Article  CAS  Google Scholar  * Lima-Costa MF, Rodrigues LC, Barreto ML _et al_: Genomic ancestry and

ethnoracial self-classification based on 5,871 community-dwelling Brazilians (The Epigen Initiative). _Sci Rep_ 2015; 5: 9812. Article  CAS  Google Scholar  * Shears SB, Gokhale NA, Wang H,

Zaremba A : Diphosphoinositol polyphosphates: what are the mechanisms? _Adv Enzyme Regul_ 2011; 51: 13–25. Article  CAS  Google Scholar  * Barker CJ, Illies C, Gaboardi GC, Berggren PO :

Inositol pyrophosphates: structure, enzymology and function. _Cell Mol Life Sci_ 2009; 66: 3851–3871. Article  CAS  Google Scholar  * Burton A, Hu X, Saiardi A : Are inositol pyrophosphates

signalling molecules? _J Cell Physiol_ 2009; 220: 8–15. Article  CAS  Google Scholar  * Hua LV, Hidaka K, Pesesse X, Barnes LD, Shears SB : Paralogous murine Nudt10 and Nudt11 genes have

differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases. _Biochem J_ 2003; 373: 81–89. Article 

CAS  Google Scholar  * Pesesse X, Choi K, Zhang T, Shears SB : Signaling by higher inositol polyphosphates. Synthesis of bisdiphosphoinositol tetrakisphosphate ('InsP8') is

selectively activated by hyperosmotic stress. _J Biol Chem_ 2004; 279: 43378–43381. Article  CAS  Google Scholar  * Song Y, Jayaraman S, Yang B, Matthay MA, Verkman AS : Role of aquaporin

water channels in airway fluid transport, humidification, and surface liquid hydration. _J Gen Physiol_ 2001; 117: 573–582. Article  CAS  Google Scholar  * Neuhofer W : Role of NFAT5 in

inflammatory disorders associated with osmotic stress. _Curr Genomics_ 2010; 11: 584–590. Article  CAS  Google Scholar  * Dobyns WB, Filauro A, Tomson BN _et al_: Inheritance of most

X-linked traits is not dominant or recessive, just X-linked. _Am J Med Genet A_ 2004; 129A: 136–143. Article  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by

the Department of Science and Technology (DECIT, Ministry of Health) and National Fund for Scientific and Technological Development (FNDCT, Ministry of Science and Technology), Funding of

Studies and Projects (FINEP, Ministry of Science and Technology, Brazil). We thank all the patients and field workers that have participated of this work. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Brazil Cintia Rodrigues Marques, Neuza Maria Alcantara-Neves & Camila Alexandrina Figueiredo *

Instituto de Saúde Coletiva, Universidade Federal da Bahia, Salvador, Brazil Gustavo NO Costa, Thiago Magalhães da Silva, Pablo Oliveira & Maurício Lima Barreto * Departamento de

Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, Jequié, Brazil Thiago Magalhães da Silva * Centro de Pesquisas Gonçalo Muniz, FIOCRUZ, Salvador, Brazil Pablo Oliveira & 

Maurício Lima Barreto * School of Medicine, ProAR-Center of Excellence in Asthma, Federal University of Bahia School of Medicine, Salvador, Brazil Alvaro A Cruz * Departamento de

Estatística, Instituto de Matemática, Universidade Federal da Bahia, Salvador, Brazil Rosemeire L Fiaccone * Departamento de Medicina Social, Programa de Pós-Graduação em Epidemiologia,

Universidade Federal de Pelotas, Pelotas, Brazil Bernardo L Horta & Fernando Pires Hartwig * Department of Medicine, University of California, San Francisco, CA, USA Esteban G Burchard *

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA Esteban G Burchard * Department of Medicine, Hospital Universitario N.S. de

Candelaria, Tenerife, Spain Maria Pino-Yanes * Department of Medicine, University of California, San Francisco (UCSF), CA, USA Maria Pino-Yanes * Department of Infectious Disease

Epidemiology, Faculty of Epidemiology, London School of Hygiene and Tropical Medicine, Laura C Rodrigues * Instituto de Pesquisa Rene Rachou, Fundação Oswaldo Cruz. Av. Augusto de Lima, Belo

Horizonte, Minas Gerais, Brazil Maria Fernanda Lima-Costa * Faculdade de Medicina, Instituto do Coração, Universidade de São Paulo, São Paulo, Brazil Alexandre C Pereira * Departamento de

Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Mateus H Gouveia, Hanaisa P Sant Anna & Eduardo

Tarazona-Santos Authors * Cintia Rodrigues Marques View author publications You can also search for this author inPubMed Google Scholar * Gustavo NO Costa View author publications You can

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Scholar CORRESPONDING AUTHOR Correspondence to Camila Alexandrina Figueiredo. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION

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association between variants in _IL1RAPL_ and asthma symptoms in Latin American children. _Eur J Hum Genet_ 25, 439–445 (2017). https://doi.org/10.1038/ejhg.2016.197 Download citation *

Received: 02 May 2016 * Revised: 22 November 2016 * Accepted: 06 December 2016 * Published: 25 January 2017 * Issue Date: April 2017 * DOI: https://doi.org/10.1038/ejhg.2016.197 SHARE THIS

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