Sheila J. Barton

The effect of genotype and in utero environment on interindividual variation in neonate DNA methylomes

Integrating the genotype with epigenetic marks holds the promise of better understanding the biology that underlies the complex interactions of inherited and environmental components that define the developmental origins of a range of disorders. The quality of the in utero environment significantly influences health over the lifecourse. Epigenetics, and in particular DNA methylation marks, have been postulated as a mechanism for the enduring effects of the prenatal environment. Accordingly, neonate methylomes contain molecular memory of the individual in utero experience. However, interindividual variation in methylation can also be a consequence of DNA sequence polymorphisms that result in methylation quantitative trait loci (methQTLs) and, potentially, the interaction between fixed genetic variation and environmental influences. We surveyed the genotypes and DNA methylomes of 237 neonates and found 1423 punctuate regions of the methylome that were highly variable across individuals, termed variably methylated regions (VMRs), against a backdrop of homogeneity. MethQTLs were readily detected in neonatal methylomes, and genotype alone best explained ∼25% of the VMRs. We found that the best explanation for 75% of VMRs was the interaction of genotype with different in utero environments, including maternal smoking, maternal depression, maternal BMI, infant birth weight, gestational age, and birth order. Our study sheds new light on the complex relationship between biological inheritance as represented by genotype and individual prenatal experience and suggests the importance of considering both fixed genetic variation and environmental factors in interpreting epigenetic variation.

TOLL-LIKE RECEPTOR 4 POLYMORPHISM AND SEVERITY OF ATOPY IN ASTHMATICS

Endotoxin exposure may have a protective effect against asthma and atopy. An Asp299Gly polymorphism in the Toll-like receptor 4 (TLR4) gene reduces responsiveness to endotoxin. This study determined the effect of TLR4 polymorphism on the risk and severity of asthma and atopy. In all, 336 UK Caucasian families with ≥2 affected sibs (physicians diagnosis of asthma and current medication use) and 179 Caucasians without asthma or a family history of asthma were genotyped using ARMS-PCR. No association of the TLR4 polymorphism was found with the risk of developing asthma, either in parent-affected sibling trios, or in case–control analyses (P>0.05). In the first affected asthmatic siblings, the atopy severity score (based on size and number of positive skin-prick tests and specific IgE) was higher in those with the Asp/Gly or Gly/Gly genotypes (mean 1.8, s.d. 1.1, n=39) compared to those with the Asp/Asp genotype (mean 1.2, s.d. 1.0, n=279) (P=0.003, t-test). No associations were found with total IgE, FEV1 % predicted, slope of FEV1 response to methacholine or asthma severity score (P>0.05). This study confirms the previously observed lack of association of TLR4 polymorphisms with asthma. In contrast, the findings suggest that genetically determined hyporesponsiveness to endotoxin may increase atopy severity.

Polymorphisms in the interleukin-4 and interleukin-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population

Background  IL‐4 by binding to its receptor (IL‐4R) is essential for the development of airway inflammation present in asthma, through the induction of IgE synthesis in B cells and differentiation of T cells to a Th2 phenotype.

Identification of PCDH1 as a novel susceptibility gene for bronchial hyperresponsiveness.

RATIONALE Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide. Asthma is caused by interaction of genetic and environmental factors. Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants. BHR and asthma are linked to chromosome 5q31-q33. OBJECTIVES To identify a gene for BHR on chromosome 5q31-q33. METHODS In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified. PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants. Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples. PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS In seven out of eight populations (n = 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05) This association was present in both families with asthma and general populations. PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages. CONCLUSIONS PCDH1 is a novel gene for BHR in adults and children. The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.

Allelic association and functional studies of promoter polymorphism in the leukotriene C4 synthase gene (LTC4S) in asthma

Background: LTC4 synthase is essential for the production of cysteinyl leukotrienes (Cys-LT), critical mediators in asthma. We have identified a novel promoter polymorphism at position −1072 (G/A) and a −444 (A/C) polymorphism has previously been reported. The role of these polymorphisms in the genetic susceptibility to asthma was examined. Methods: To test for genetic association with asthma phenotypes, 341 white families (two asthmatic siblings) and 184 non-asthmatic control subjects were genotyped. Genetic association was assessed using case control and transmission disequilibrium test (TDT) analyses. LTC4S promoter luciferase constructs and transiently transfected human HeLa and KU812F cells were generated to determine the functional role of these polymorphisms on basal transcription. Results: No associations were observed in case control analyses (–1072 A, q=0.09; −444 C, q=0.29); the TDT identified a borderline association between the −444 C allele and bronchial responsiveness to methacholine (p=0.065). Asthmatic children with the −444 C allele had a lower mean basal forced expiratory volume in 1 second (97.4 v 92.7% predicted, p=0.005). LTC4S promoter luciferase analyses provided no evidence for a functional role of either polymorphism in determining basal transcription. Conclusion: This study does not support a role for these polymorphisms in genetic susceptibility to asthma but provides evidence to suggest a role in determining lung function parameters.

Promoter polymorphism in the 5-lipoxygenase (ALOX5) and 5-lipoxygenase-activating protein (ALOX5AP) genes and asthma susceptibility in a Caucasian population

Background 5‐Lipoxygenase (5‐LO) and 5‐lipoxygenase‐activating protein (FLAP) are essential for cysteinyl‐leukotriene (cys‐LT) production, critical mediators in asthma.

Lack of association of HLA class I genes and TNF alpha-308 polymorphism in toluene diisocyanate-induced asthma

Background:  Toluene diisocyanate (TDI)‐induced asthma is a common cause of occupational asthma and it affects 5–15% of the exposed population suggesting an underlying genetic susceptibility.

Topical intranasal corticosteroids in 4-11 year old children with persistent bilateral otitis media with effusion in primary care: double blind randomised placebo controlled trial

Objective To determine the clinical effectiveness of topical intranasal corticosteroids in children with bilateral otitis media with effusion. Design Double blind randomised placebo controlled trial. Setting 76 Medical Research Council General Practice Research Framework practices throughout the United Kingdom, between 2004 and 2007. Participants 217 children aged 4-11 years who had at least one practice recorded episode of otitis media or a related ear problem in the previous 12 months, and with bilateral otitis media with effusion confirmed by a research nurse using otoscopy plus micro-tympanometry (B/B or B/C2, modified Jerger types). Intervention Mometasone furoate 50 µg or placebo spray given once daily into each nostril for three months. Main outcome measures Proportions of children cured of bilateral otitis media with effusion assessed with tympanometry (C1 or A type) at one month (primary end point), three months, and nine months; adverse events; three month diary symptoms. Results 41% (39/96) of the topical steroid group and 45% (44/98) of the placebo group were cured in one or both ears at one month (difference favouring placebo 4.3% (95% confidence interval −9.3% to 18.1%). Poisson regression was done with adjustment for four pre-specified covariates (clinical severity, P=0.003; atopy, P=0.67; age, P=0.92; season, P=0.71). The adjusted relative risk at one month was 0.97 (95% confidence interval 0.74 to 1.26). At three months, 58% of the topical steroid group and 52% of the placebo group were cured (relative risk 1.23, 0.84 to 1.80). Diary symptoms did not differ between the two groups, and no significant harms were reported. Conclusions Topical steroids are unlikely to be an effective treatment for otitis media with effusion in general practice. High rates of natural resolution occurred by 1-3 months. Trial registration Current Controlled Trials ISRCTN38988331; National Research Register NO575123823; MREC 03/11/073.

Identification of Lipocalin and Apolipoprotein A1 as Biomarkers of Chronic Obstructive Pulmonary Disease

RATIONALE Much effort is being made to discover noninvasive biomarkers of chronic airway disease that might enable better management, predict prognosis, and provide new therapeutic targets. OBJECTIVES To undertake a comprehensive, unbiased proteomic analysis of induced sputum and identify novel noninvasive biomarkers for chronic obstructive pulmonary disease (COPD). METHODS Induced sputum was obtained from patients with COPD with a spectrum of disease severity and from control subjects. Two-dimensional gel electrophoresis and mass spectrometric identification of differentially expressed proteins were first applied to induced sputum from patients with GOLD stage 2 COPD and healthy smoker control subjects. Initial results thus obtained were validated by a combination of immunoassays (Western blotting and ELISA) applied to a large subject cohort. The biomarkers were localized to bronchial mucosa by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS Of 1,325 individual protein spots identified, 37 were quantitatively and 3 qualitatively different between the two groups (P < 0.05%). Forty protein spots were subjected to tandem mass spectrometry, which identified 15 separate protein species. Seven of these were further quantified in induced sputum from 97 individuals. Using this sequential approach, two of these potential biomarkers (apolipoprotein A1 and lipocalin-1) were found to be significantly reduced in patients with COPD when compared with healthy smokers. Their levels correlated with FEV(1)/FVC, indicating their relationship to disease severity. CONCLUSIONS A potential role for apolipoprotein A1 and lipocalin-1 in innate defense has been postulated previously; our discovery of their reduction in COPD indicates a deficient innate defense system in airway disease that could explain increased susceptibility to infectious exacerbations.

The role of LTA4H and ALOX5AP polymorphism in asthma and allergy susceptibility

Background:  Leukotrienes (LTs) have been identified as central mediators in asthma and allergy. Pharmacological inhibition of cysteinyl‐LT activity improves asthma symptoms and control. Accumulating evidence suggests a role for the dihydroxy leukotriene LTB4 in airway disease. LTA4 hydrolase and 5‐lipoxygenase activating protein have key roles in LTB4 production. Single nucleotide polymorphism (SNPs) and haplotypes spanning the LTA4H and ALOX5AP genes have been associated with LTB4 production and myocardial infarction (MI).