The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma.
We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance.
PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding.
In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair.
The leptin gene (LEP) plays a regulatory role in satiety, inflammation, and allergy. Prior findings linking leptin to asthma motivated us to investigate whether DNA methylation (DNA-M) of CpG (cytosine-phosphate-guanine) sites in concert with single nucleotide polymorphisms (SNPs) of LEP can explain the risk of asthma and lung function. Methylation of CpG sites was assessed using the Illumina Infinium Human Methylation 450 beadchip in blood samples collected from 10- and 18-year-old boys and girls from the Isle of Wight (IOW) birth cohort (UK). Four LEP SNPs were genotyped. Linear and log linear models were used for the analysis, adjusting for false discovery rate (FDR). The analyses were repeated in the BAMSE cohort (Sweden). In the IOW study, the interaction of cg00666422 and rs11763517 (CT vs TT and CC) was associated with FEV1 (FDR-adjusted p-value: 0.03), FEV1/FVC ratio (FDR-adjusted p-value: 0.0096), and FEF25-75% (FDR-adjusted p-value: 0.00048) such that they decreased with increasing DNA-M. The interaction of the same CpG-SNP pair was also associated with increased risk of asthma at age 18. We replicated the findings for FEV1/FVC and FEF25-75% in a smaller sample of 34 participants at age 10. Regarding the BAMSE cohort, although, the interaction of cg00666422 and rs11763517 on lung function were not significant, the direction of the effect was the same as in IOW cohort. Thus, penetrance of LEP genotype seems to be modified by methylation at cg00666422 and is linked to airway obstruction and asthma.
Epigenetics; CpGs; SNPs; spirometry; FEV1; FEV1/FVC ratio; FEF25-75%; asthma
The FTO gene harbors variation with the strongest effect on adiposity and obesity risk. Previous data support a role for FTO variation in influencing food intake. We conducted a combined analysis of 16,094 boys and girls aged 1–18 years from 14 studies to examine the following: 1) the association between the FTO rs9939609 variant (or a proxy) and total energy and macronutrient intake; and 2) the interaction between the FTO variant and dietary intake, and the effect on BMI. We found that the BMI-increasing allele (minor allele) of the FTO variant was associated with increased total energy intake (effect per allele = 14.3 kcal/day [95% CI 5.9, 22.7 kcal/day], P = 6.5 × 10−4), but not with protein, carbohydrate, or fat intake. We also found that protein intake modified the association between the FTO variant and BMI (interactive effect per allele = 0.08 SD [0.03, 0.12 SD], P for interaction = 7.2 × 10−4): the association between FTO genotype and BMI was much stronger in individuals with high protein intake (effect per allele = 0.10 SD [0.07, 0.13 SD], P = 8.2 × 10−10) than in those with low intake (effect per allele = 0.04 SD [0.01, 0.07 SD], P = 0.02). Our results suggest that the FTO variant that confers a predisposition to higher BMI is associated with higher total energy intake, and that lower dietary protein intake attenuates the association between FTO genotype and adiposity in children and adolescents.
Prenatal and infant acetaminophen exposure has been associated with an increased risk of childhood asthma phenotypes. Demonstration of biologically plausible interactions between these exposures and maternal and child antioxidant gene polymorphisms would strengthen causal inference.
To explore potential interactions between prenatal and infant acetaminophen exposure and antioxidant genotypes on childhood asthma.
In the Avon Longitudinal Study of Parents and Children (ALSPAC) we typed a functional Nuclear erythroid 2 p45-related factor 2 (Nrf2) polymorphism and glutathione S-transferase (GST) M1, T1 and P1 polymorphisms. Effects of prenatal and infant acetaminophen exposure on asthma phenotypes at 7 years were stratified by genotype in >4,000 mothers and >5,000 children.
Risk of asthma and wheezing associated with early gestation acetaminophen exposure was increased when maternal copies of the minor T allele of Nrf2 were present (P interaction 0.02 and 0.04, respectively). Risk of asthma associated with late gestation exposure was higher when maternal GSTT1 genotype was present, rather than absent (P interaction 0.006), and risk of wheezing was increased when maternal GSTM1 was present (P interaction 0.04). Whilst acetaminophen use in infancy was associated with an increased risk of atopy, child antioxidant genotype did not modify associations between infant acetaminophen use and asthma phenotypes. However, the increased risk of asthma and wheezing associated with late gestation acetaminophen exposure in the presence of maternal GSTM1 was further enhanced when GSTM1 was also present in the child.
Maternal antioxidant gene polymorphisms may modify the relation between prenatal acetaminophen exposure and childhood asthma, strengthening evidence for a causal association. In contrast, relations between infant acetaminophen use and asthma and atopy were not modified by child genotype, and may be confounded by pre-existing wheeze or allergy.
Asthma; Acetaminophen; paracetamol; Glutathione-S-transferase; Nrf2; Prenatal Exposure; Delayed Effects; ALSPAC; pregnancy; birth cohort; genotype; gene environment interaction
Several regions of the genome have shown to be associated with COPD in genome-wide association studies of common variants.
To determine rare and potentially functional single nucleotide polymorphisms (SNPs) associated with the risk of COPD and severity of airflow limitation.
3226 current or former smokers of European ancestry with lung function measures indicative of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2 COPD or worse were genotyped using an exome array. An analysis of risk of COPD was carried out using ever smoking controls (n=4784). Associations with %predicted FEV1 were tested in cases. We followed-up signals of interest (p<10−5) in independent samples from a subset of the UK Biobank population and also undertook a more powerful discovery study by meta-analysing the exome array data and UK Biobank data for variants represented on both arrays.
Among the associated variants were two in regions previously unreported for COPD; a low frequency non-synonymous SNP in MOCS3 (rs7269297, pdiscovery=3.08×10−6, preplication=0.019) and a rare SNP in IFIT3, which emerged in the meta-analysis (rs140549288, pmeta=8.56×10−6). In the meta-analysis of % predicted FEV1 in cases, the strongest association was shown for a splice variant in a previously unreported region, SERPINA12 (rs140198372, pmeta=5.72×10−6). We also confirmed previously reported associations with COPD risk at MMP12, HHIP, GPR126 and CHRNA5. No associations in novel regions reached a stringent exome-wide significance threshold (p<3.7×10−7).
This study identified several associations with the risk of COPD and severity of airflow limitation, including novel regions MOCS3, IFIT3 and SERPINA12, which warrant further study.
COPD epidemiology; Tobacco and the lung
Genetic association studies have identified 21 loci associated with atopic dermatitis risk predominantly in populations of European ancestry. To identify further susceptibility loci for this common complex skin disease, we performed a meta-analysis of >15 million genetic variants in 21,399 cases and 95,464 controls from populations of European, African, Japanese and Latino ancestry, followed by replication in 32,059 cases and 228,628 controls from 18 studies. We identified 10 novel risk loci, bringing the total number of known atopic dermatitis risk loci to 31 (with novel secondary signals at 4 of these). Notably, the new loci include candidate genes with roles in regulation of innate host defenses and T-cell function, underscoring the important contribution of (auto-)immune mechanisms to atopic dermatitis pathogenesis.
Nonbiased approaches, especially genome wide association studies (GWAS) have identified novel molecular targets in the pathogenesis of asthma, but so far only account for a small proportion of the heritability of asthma. Recognition of the importance of disease heterogeneity, the need for improved disease phenotyping and that genes involved in the inception of asthma are likely to be different from those involved in severity widens the impact of asthma genetics. Genes implicated in multiple causal pathways identifies the use of genetic scores to capture the impact of genetic variations on individuals. Gene-environmental interaction adds another layer of complexity which is being successfully explored by epigenetic approaches. Pharmacogenetics is one example of gene-environment interaction which is already having application in determining drug responders from non-responders and those most susceptible to adverse effects. Such applications represent one aspect of personalised medicine designed to help place the individual at the centre of healthcare.
FTO is the strongest known genetic susceptibility locus for obesity. Experimental studies in animals suggest the potential roles of FTO in regulating food intake. The interactive relation among FTO variants, dietary intake and body mass index (BMI) is complex and results from previous often small-scale studies in humans are highly inconsistent. We performed large-scale analyses based on data from 177 330 adults (154 439 Whites, 5776 African Americans and 17 115 Asians) from 40 studies to examine: (i) the association between the FTO-rs9939609 variant (or a proxy single-nucleotide polymorphism) and total energy and macronutrient intake and (ii) the interaction between the FTO variant and dietary intake on BMI. The minor allele (A-allele) of the FTO-rs9939609 variant was associated with higher BMI in Whites (effect per allele = 0.34 [0.31, 0.37] kg/m2, P = 1.9 × 10−105), and all participants (0.30 [0.30, 0.35] kg/m2, P = 3.6 × 10−107). The BMI-increasing allele of the FTO variant showed a significant association with higher dietary protein intake (effect per allele = 0.08 [0.06, 0.10] %, P = 2.4 × 10−16), and relative weak associations with lower total energy intake (−6.4 [−10.1, −2.6] kcal/day, P = 0.001) and lower dietary carbohydrate intake (−0.07 [−0.11, −0.02] %, P = 0.004). The associations with protein (P = 7.5 × 10−9) and total energy (P = 0.002) were attenuated but remained significant after adjustment for BMI. We did not find significant interactions between the FTO variant and dietary intake of total energy, protein, carbohydrate or fat on BMI. Our findings suggest a positive association between the BMI-increasing allele of FTO variant and higher dietary protein intake and offer insight into potential link between FTO, dietary protein intake and adiposity.
Filaggrin is an epidermal protein that has a role in skin barrier function. Filaggrin loss-of-function FLG-LOF) mutations are a significant risk factor for eczema and atopy, but their association with food allergy (FA) is less clear.
We explored the longitudinal relationship between three common FLG-LOF mutations and FA using the Isle of Wight birth cohort.
FA diagnosis was based on recognised allergic reactions within 4 hours following exposure to known food allergens. Food allergen sensitization (FAS) was identified by skin prick test conducted between 1–18 years to a range of food allergens. Three filaggrin mutations were genotyped in 1150/1456 children (79%). The temporal relationships between FA, FAS and eczema in children with filaggrin mutations were explored using path analysis with total, direct and indirect effect models.
There was a significant total effect of FLG-LOF mutations on the risk of FA in later childhood at ages 10 (OR: 31.46, 95%CI 2.86, >100) and 18 years (OR: 4.25, 95%CI 1.55, 11.61). Path analysis showed that there was no direct effect of FLG-LOF mutations on FA at any age, however an indirect effect was found on FA at all ages via eczema and FAS in the earlier years
FLG-LOF mutations are associated with FA in older children via eczema and FAS in their early childhood. Our results highlight a biologically plausible pathway, which suggests that skin barrier function is important in the development and persistence of FA.
Food Allergy; Filaggrin; FLG-LOF; Food allergen sensitization; Path analysis; Prediction; Eczema
Associations of filaggrin (FLG) variants with asthma and
rhinitis have been shown to be modulated by eczema status. However, it is unknown
whether allergic sensitization status modifies this association. The aim of this study
was to determine whether FLG variants need eczema and/or allergic
sensitization as a necessary component to execute its adverse effect on coexisting and
subsequent asthma and rhinitis.
Repeated measurements of asthma, rhinitis, eczema, and allergic sensitization
(documented by skin prick tests) at ages 1, 2, 4, 10, and 18 years were ascertained in
the Isle of Wight birth cohort (n = 1,456). FLG
haploinsufficiency was defined as having at least the minor allele of R501X, 2282del4,
or S3247X variants. Log binomial regression models were used to test associations and
FLG variants increased the risk of asthma (RR = 1.39,
95% CI: 1.06 – 1.80) and rhinitis (RR = 1.37, 95% CI:
1.16 – 1.63). In delayed effect models, ‘FLG variants
plus allergic sensitization’ and ‘FLG variants plus
eczema’ increased the risk of subsequent asthma by 4.93-fold (95% CI:
3.61 – 6.71) and 3.33-fold (95% CI: 2.45 – 4.51), respectively,
during the first 18 years of life. In contrast, neither eczema nor allergic
sensitization in combination with FLG variants increased the risk of
Allergic sensitization and eczema modulated the association between
FLG variants and asthma, but not rhinitis. Results of our study imply
that the mechanisms and pathways through which FLG variants predispose
to increased risk of asthma and rhinitis may be different.
filaggrin variants; asthma; rhinitis; allergic sensitization; eczema; interaction
Allergic sensitization and filaggrin gene (FLG) variants are important risk factors for allergic disorders; however, knowledge on their individual and interactive effects on the coexistence of eczema, asthma, and rhinitis is lacking.
This study aimed at investigating the single and combined effects of allergic sensitization and FLG variants on the development of single and multiple allergic disorders.
The Isle of Wight Birth Cohort (n = 1,456) has been examined at 1, 2, 4, 10, and 18 years of age. Repeated measurements of eczema, asthma, rhinitis, and skin prick tests were available for all follow-ups. FLG variants were genotyped in 1,150 participants. Associations of allergic sensitization and FLG variants with single and multiple allergic disorders were tested in log-binomial regression analysis.
The prevalence of eczema-, asthma-, and rhinitis-only ranged from 5.6% to 8.5%, 4.9% to 10.2%, and 2.5% to 20.4%, respectively, during the first 18 years of life. The coexistence of allergic disorders is common, with approximately 2% of the population reporting the comorbidity of “eczema, asthma, and rhinitis” during the study period. In repeated measurement analyses, allergic sensitization and FLG variants, when analyzed separately, were associated with having single and multiple allergic disorders. Of particular significance, their combined effect increased the risk of “eczema and asthma” (RR = 13.67, 95% CI: 7.35 – 25.42), “asthma and rhinitis” (RR = 7.46, 95% CI: 5.07 – 10.98), and “eczema, asthma, and rhinitis” (RR = 23.44, 95% CI: 12.27 – 44.78).
Conclusions and Clinical Relevance
The coexistence of allergic disorders is frequent and allergic sensitization and FLG variants jointly increased risk of allergic comorbidities, which may represent more severe and complex clinical phenotypes. The interactive effect and the elevated proportion of allergic comorbidities associated with allergic sensitization and FLG variants emphasize their joint importance in the pathogenesis of allergic disorders.
The prevalence of allergic diseases are increasing worldwide, emphasizing the need to elucidate their pathogeneses. The aims of this study were to use a two-stage design to identify DNA methylation levels at cytosine–phosphate–guanine (CpG) sites across the genome associated with atopy and high serum immunoglobulin E (IgE), then to replicate our findings in an independent cohort.
Atopy was assessed via skin prick tests and high serum IgE. Methylation levels were measured from whole blood using the Illumina Infinium HumanMethylation450 BeadChip from 18-year-old women (n = 245) and men (n = 122) in the Isle of Wight birth cohort. After data cleaning and processing, and removing probes with possible single nucleotide polymorphisms, DNA methylation levels from 254,460 CpG sites from the 245 women were subjected to recursive Random Forest feature selection for stage 1. The sites selected from stage 1 were tested in stage 2 for associations with atopy and high IgE levels (>200 kU/L) via logistic regression adjusted for predicted cell-type proportions and sex. Sites significantly associated with atopy in stage 2 underwent replication tests in the independent Swedish birth cohort BAMSE (n = 464).
In stage 1, 62 sites were selected, of which 22 were associated with atopy in stage 2 (P-value range 6.5E−9 to 1.4E−5) and 12 associated with high IgE levels (P-value range 1.1E−5 to 7.1E−4) at the Bonferroni adjusted alpha (0.05/62 = 0.0008). Of the 19 available sites, 13 were replicated.
We identified 13 novel epigenetic loci associated with atopy and high IgE that could serve as candidate loci for future studies; four were within genes with known roles in the immune response (cg04983687 in the body of ZFPM1, cg18219873 in the 5′UTR of PRG2, cg27469152 in the 3′UTR of EPX, and cg09332506 in the body of COPA).
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0213-8) contains supplementary material, which is available to authorized users.
The Illumina Infinium HumanMethylation450 BeadChip is an array-based technology for analysing DNA methylation at approximately 475,000 differentially methylated cytosines across the human genome. Hitherto, the array has been used for case-control studies, where sample numbers can be sufficient to yield statistically robust data on a genome-wide basis. We recently reported an informatic pipeline capable of yielding statistically and biologically significant results using only five cases, which expanded the use of this technology to rare disease studies. However, the clinical application of these technologies requires the ability to perform robust analysis of individual patients.
Here we report a novel informatic approach for methylation array analysis of single samples, using the Crawford-Howell t-test. We tested our approach on patients with ultra-rare imprinting disorders with aberrant DNA methylation at multiple locations across the genome, which was previously detected by targeted testing. However, array analysis outperformed targeted assays in three ways: it detected loci not normally analysed by targeted testing, detected methylation changes too subtle to detect by the targeted testing and reported broad and consistent methylation changes across genetic loci not captured by point testing.
This method has potential clinical utility for human disorders where DNA methylation change may be a biomarker of disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0081-5) contains supplementary material, which is available to authorized users.
Methylation; Illumina HumanMethylation450 array; Single case-control analysis; Crawford-Howell t-test
Phthalates may increase the asthma risk in children. Mechanisms underlying this association remain to be addressed. This study assesses the effect of phthalate exposures on epigenetic changes and the role of epigenetic changes for asthma. In the first step, urine and blood samples from 256 children of the Childhood Environment and Allergic diseases Study (CEAS) were analyzed. Urine 5OH-MEHP levels were quantified as an indicator of exposure, and asthma information was collected. DNA methylation (DNA-M) was measured by quantitative PCR. In the screening part of step 1, DNA-M of 21 potential human candidate genes suggested by a toxicogenomic data were investigated in 22 blood samples. Then, in the testing part of step 1, positively screened genes were tested in a larger sample of 256 children and then validated by protein measurements. In step 2, we replicated the association between phthalate exposure and gene-specific DNA-M in 54 children in the phthalate contaminated food event. In step 3, the risk of DNA-M for asthma was tested in 256 children from CEAS and corroborated in 270 children from the Isle of Wight (IOW) birth cohort.
Differential methylation in three genes (AR, TNFα, and IL-4) was identified through screening. Testing in 256 children showed that methylation of the TNFα gene promoter was lower when children had higher urine 5OH-MEHP values (β = −0.138, P = 0.040). Functional validation revealed that TNFα methylation was inversely correlated with TNFα protein levels (β = −0.18, P = 0.041). In an additional sample of 54 children, we corroborated that methylation of the TNFα gene promoter was lower when urine 5OH-MEHP concentrations were higher. Finally, we found that a lower methylation of 5′CGI region of TNFα was associated with asthma in 256 CEAS children (OR = 2.15, 95% CI = 1.01 to 4.62). We replicated this in 270 children from the IOW birth cohort study. Methylation of the CpG site cg10717214 was negatively associated with asthma, when children had ‘AA’ or ‘AG’ genotype of the TNFα single nucleotide rs1800610.
Effects of phthalate exposure on asthma may be mediated through alterations in DNA methylation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0060-x) contains supplementary material, which is available to authorized users.
DEHP exposure; DNA methylation; TNFα; Asthma; Quantitative PCR
Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 × 10−6) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; β = 0.046, SE = 0.008, P = 2.46 × 10−8, explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 × 10−4) and adult height (N = 127 513; P = 1.45 × 10−5). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
Allergic disease development is affected by both genes and the environment, and epigenetic mechanisms are hypothesized to mediate these environmental effects. In this article, we discuss the link between the environment, DNA methylation and allergic disease, as well as questions of causality inherent to analyses of DNA methylation. From the practical side, we describe characteristics of allergic phenotypes and contrast different epidemiologic study designs used in epigenetic research. We examine methodological considerations, how best to conduct preprocessing and analysis of DNA methylation data sets, and the latest methods, technologies and discoveries in this rapidly advancing field. DNA methylation and other epigenetic marks are firmly entwined with allergic disease, a link that may hold the basis for future allergic disease diagnosis and treatment.
allergy; asthma; DNA methylation; environment; epigenetic
The effectiveness of intermittent montelukast for wheeze in young children is unclear. We aimed to assess whether intermittent montelukast is better than placebo for treatment of wheeze in this age group. Because copy numbers of the Sp1-binding motif in the arachidonate 5-lipoxygenase (ALOX5) gene promoter (either 5/5, 5/x, or x/x, where x does not equal 5) modifies response to montelukast in adults, we stratified by this genotype.
We did this multicentre, parallel-group, randomised, placebo-controlled trial between Oct 1, 2010, and Dec 20, 2013, at 21 primary care sites and 41 secondary care sites in England and Scotland. Children aged 10 months to 5 years with two or more wheeze episodes were allocated to either a 5/5 or 5/x+x/x ALOX5 promoter genotype stratum, then randomly assigned (1:1) via a permuted block schedule (size ten), to receive intermittent montelukast or placebo given by parents at each wheeze episode over a 12 month period. Clinical investigators and parents were masked to treatment group and genotype strata. The primary outcome was number of unscheduled medical attendances for wheezing episodes. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01142505.
We randomly assigned 1358 children to receive montelukast (n=669) or placebo (n=677). Consent was withdrawn for 12 (1%) children. Primary outcome data were available for 1308 (96%) children. There was no difference in unscheduled medical attendances for wheezing episodes between children in the montelukast and placebo groups (mean 2·0 [SD 2·6] vs 2·3 [2·7]; incidence rate ratio [IRR] 0·88, 95% CI: 0·77–1·01; p=0·06). Compared with placebo, unscheduled medical attendances for wheezing episodes were reduced in children given montelukast in the 5/5 stratum (2·0 [2·7] vs 2·4 [3·0]; IRR 0·80, 95% CI 0·68–0·95; p=0·01), but not in those in the 5/x+x/x stratum (2·0 [2·5] vs 2·0 [2·3]; 1·03, 0·83–1·29; p=0·79, pinteraction=0·08). We recorded one serious adverse event, which was a skin reaction in a child allocated to placebo.
Our findings show no clear benefit of intermittent montelukast in young children with wheeze. However, the 5/5 ALOX5 promoter genotype might identify a montelukast-responsive subgroup.
Medical Research Council (UK) and National Institute for Health Research.
The prevalence of asthma in girls increases after puberty. Previous studies have detected associations between sex hormones and asthma, as well as between sex hormones and T helper 2 (Th2) asthma-typical immune responses. Therefore, we hypothesized that exogenous or endogenous sex hormone exposure (represented by oral contraceptive pill (OCP) use and early menarche, respectively) are associated with DNA methylation (DNA-M) of the Th2 transcription factor gene, GATA3, in turn affecting the risk of asthma in girls, possibly in interaction with genetic variants.
Blood samples were collected from 245 female participants aged 18 years randomly selected for methylation analysis from the Isle of Wight birth cohort, UK. Information on use of OCPs, age at menarche, and concurrent asthma were assessed by questionnaire. Genome-wide DNA-M was determined using the Illumina Infinium HumanMethylation450 beadchip. In a first stage, we tested the interaction between sex hormone exposure and genetic variants on DNA-M of specific cytosine-phosphate-guanine (CpG) sites. In a second stage, we determined whether these CpG sites interact with genetic variants in GATA3 to explain the risk of asthma.
Interactions between OCP use and seven single nucleotide polymorphisms (SNPs) of GATA3 were analyzed for 14 CpG sites (stage 1). The interaction between OCP use and SNP rs1269486 was found to be associated with the methylation level of cg17124583 (P = 0.002, false discovery rate (FDR) adjusted P = 0.04). DNA-M of this same CpG site was also influenced by the interaction between age at menarche and rs1269486 (P = 0.0017). In stage 2, we found that cg17124583 modified the association of SNP rs422628 with asthma risk at the age of 18 years (P = 0.006, FDR adjusted P = 0.04). Subjects with genotype AG showed an increase in average risk ratio (RR) from 0.31 (95% CI: 0.10 to 0.8) to 11.65 (95% CI: 1.71 to 79.5) when methylation level increased from 0.02 to 0.12, relative to genotype AA.
A two-stage model consisting of genetic variants in the GATA3 gene, OCP use, age at menarche, and DNA-M may explain how sex hormones in women can increase the asthma prevalence after puberty.
GATA3 gene; DNA methylation; genetic variants; epigenetics; oral contraceptives; age at menarche; asthma; puberty; adolescence; single nucleotide polymorphism; CpG
Chronic cardiorespiratory disease is associated with low birthweight suggesting the importance of the developmental environment. Prenatal factors affecting fetal growth are believed important, but the underlying mechanisms are unknown. The influence of developmental programming on bronchial hyperreactivity is investigated in an animal model and evidence for comparable associations is sought in humans. Pregnant Wistar rats were fed either control or protein-restricted diets throughout pregnancy. Bronchoconstrictor responses were recorded from offspring bronchial segments. Morphometric analysis of paraffin-embedded lung sections was conducted. In a human mother-child cohort ultrasound measurements of fetal growth were related to bronchial hyperreactivity, measured at age six years using methacholine. Protein-restricted rats' offspring demonstrated greater bronchoconstriction than controls. Airway structure was not altered. Children with lesser abdominal circumference growth during 11–19 weeks' gestation had greater bronchial hyperreactivity than those with more rapid abdominal growth. Imbalanced maternal nutrition during pregnancy results in offspring bronchial hyperreactivity. Prenatal environmental influences might play a comparable role in humans.
Genetic effects on asthma of genes in the T-helper 2 (Th2) pathway may interact with epigenetic factors including DNA methylation. We hypothesized that interactions between genetic variants and methylation in genes in this pathway (IL4, IL4R, IL13, GATA3, and STAT6) influence asthma risk, that such influences are age-dependent, and that methylation of some CpG sites changes over time in accordance with asthma transition. We tested these hypotheses in subsamples of girls from a population-based birth cohort established on the Isle of Wight, UK, in 1989.
Logistic regression models were applied to test the interaction effect of DNA methylation and SNP on asthma within each of the five genes. Bootstrapping was used to assess the models identified. From 1,361 models fitted at each age of 10 and 18 years, 8 models, including 4 CpGs and 8 SNPs, showed potential associations with asthma risk. Of the 4 CpGs, methylation of cg26937798 (IL4R) and cg23943829 (IL4) changes between ages 10 and 18 (both higher at 10; P = 9.14 × 10−6 and 1.07 × 10−5, respectively).
At age 10, the odds of asthma tended to decrease as cg12405139 (GATA3) methylation increased (log-OR = −12.15; P = 0.049); this effect disappeared by age 18. At age 18, methylation of cg09791102 (IL4R) was associated with higher risk of asthma among subjects with genotype GG compared to AG (P = 0.003), increased cg26937798 methylation among subjects with rs3024685 (IL4R) genotype AA (P = 0.003) or rs8832 (IL4R) genotype GG (P = 0.01) was associated with a lower asthma risk; these CpGs had no effect at age 10. Increasing cg26937798 methylation over time possibly reduced the risk of positive asthma transition (asthma-free at age 10 → asthma at age 18; log-OR = −3.11; P = 0.069) and increased the likelihood of negative transition (asthma at age 10 → asthma-free at age 18; log-OR = 3.97; P = 0.074).
The interaction of DNA methylation and SNPs in Th2 pathway genes is likely to contribute to asthma risk. This effect may vary with age. Methylation of some CpGs changed over time, which may influence asthma transition.
Asthma risk; Asthma transition; DNA methylation and SNP interaction; Th2 pathway
Purpose of the review
Epigenetic mechanisms have the ability to alter the phenotype without changing the genetic code. The science of epigenetics has grown considerably in recent years, and future epigenetically-based treatments or prevention strategies are likely. Epigenetic associations with asthma have received growing interest because genetic and environmental factors have been unable to independently explain the etiology of asthma.
Recent findings suggest that both the environment and underlying genetic sequence variation influence DNA methylation, which in turn seems to modify the risk conferred by genetic variants for various asthma phenotypes. In particular DNA methylation may act as an archive of a variety of early developmental exposures which then can modify the risk related to genetic variants.
Current asthma treatments may control the symptoms of asthma but do not modify its natural history. Epigenetic mechanisms and novel explanatory models provide burgeoning approaches to significantly increase our understanding of the initiation and progression of asthma. This will lead to critical information to prevent or treat asthma not only in the current generation, but due to the epigenetic inheritance may also prevent asthma in future generations.
Asthma; Epigenetics; DNA methylation; methylation quantitative trait loci; modifiable genetic variants
In 1995 the Tucson Children’s Respiratory Study (TCRS) identified clinically distinct phenotypes amongst early wheezers; the Avon Longitudinal Study of Parents And Children (ALSPAC) has recently re-examined these.
To validate statistically derived ALSPAC phenotypes in the Southampton Women’s Survey (SWS) using infant and 6 year lung function, and allergic sensitisation at 1, 3 and 6 years, comparing these with TCRS phenotypes.
Complete 6 year follow-up data were available for 926 children, selected from 1973 infants born to 12,579 women characterised pre-conception. 95 children had V’maxFRC and FEV0.4 measured age 5-14 weeks using rapid compression/raised volume techniques. At 6 years we performed spirometry (n=791), fractional exhaled nitric oxide (FeNO, n=589) and methacholine challenge (n=234). Skin prick testing was performed at 12m, 3 and 6 years (n=1494, 1255, 699, respectively). Using wheeze status questionnaire data at 6m, 12m, 2, 3 and 6 years we classified children into TCRS (never, transient early, persistent, late-onset) and ALSPAC based groups (never, early, transient, intermediate-onset, late-onset, persistent).
Amongst ALSPAC groups, persistent and late-onset wheeze were associated with atopy at 3 and 6 years, whilst intermediate-onset wheeze showed earlier atopic association at 1 year; all three were associated with FeNO at 6 years. Persistent wheezers had lower infant (V’maxFRC p<0.05) and 6 year lung function (FEV1, FEV1/FVC and FEF25-75, p<0.05), whilst late and intermediate-onset wheezers showed no lung function deficits. Transient wheezers were non-atopic but showed persistent lung function deficits (V’maxFRC in infancy, FEV1 and FEF25-75 at 6 years, all p<0.05). Those who wheezed only in the first year (early phenotype) showed no lung function deficits. No associations were seen with 6 years bronchial hyper-responsiveness or infancy FEV0.4.
SWS cohort data validates the statistically derived ALSPAC 6-class model. In particular, lung function and atopy successfully differentiate persistent, late-onset and intermediate-onset wheeze, whilst the Tucson ‘transient early’ wheeze phenotype can be sub-classified into groups that reflect early lung function. Since the 4-class model fails to adequately differentiate phenotypes based on lung function and atopy, we propose that strong consideration be given to using the 6-class paradigm for longitudinal outcome work in wheezing with onset in early life.
Wheeze; asthma; phenotype; lung function; cohort; atopy
Alcohol consumption in western pregnant women is not uncommon and could be a risk factor for childhood atopic disease. However, reported alcohol intake may be unreliable, and associations are likely to be confounded.
We aimed to study the relation between prenatal alcohol exposure and atopic phenotypes in a large population-based birth cohort with the use of a Mendelian randomization approach to minimize bias and confounding.
In white mothers and children in the Avon Longitudinal Study of Parents and Children (ALSPAC) we first analyzed associations between reported maternal alcohol consumption during pregnancy and atopic outcomes in the offspring measured at 7 years of age (asthma, wheezing, hay fever, eczema, atopy, and total IgE). We then analyzed the relation of maternal alcohol dehydrogenase (ADH)1B genotype (rs1229984) with these outcomes (the A allele is associated with faster metabolism and reduced alcohol consumption and, among drinkers, would be expected to reduce fetal exposure to ethanol).
After controlling for confounders, reported maternal drinking in late pregnancy was negatively associated with childhood asthma and hay fever (adjusted odds ratio [OR] per category increase in intake: 0.91 [95% CI, 0.82-1.01] and 0.87 [95% CI, 0.78-0.98], respectively). However, maternal ADH1B genotype was not associated with asthma comparing carriers of A allele with persons homozygous for G allele (OR, 0.98 [95% CI, 0.66-1.47]) or hay fever (OR, 1.11 [95% CI, 0.71-1.72]), nor with any other atopic outcome.
We have found no evidence to suggest that prenatal alcohol exposure increases the risk of asthma or atopy in childhood.
Alcohol; ADH1B; Mendelian randomization; prenatal exposure; ALSPAC; pregnancy; birth cohort; asthma; atopy; ADH, Alcohol dehydrogenase; ALSPAC, Avon Longitudinal Study of Parents and Children (ALSPAC); GWAS, Genome Wide Association Study; PCA, Principal Components Analysis