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1.  Epigenetic mechanisms and models in the origins of asthma 
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
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.
Summary
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.
doi:10.1097/ACI.0b013e32835ad0e7
PMCID: PMC3952069  PMID: 23242116
Asthma; Epigenetics; DNA methylation; methylation quantitative trait loci; modifiable genetic variants
2.  Validation of novel wheeze phenotypes using longitudinal airway function and atopic sensitisation data in the first 6 years of life: Evidence from the Southampton Women’s Survey. 
Pediatric pulmonology  2013;48(7):683-692.
Background
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.
Objectives
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.
Methods
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).
Results
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.
Conclusion
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.
doi:10.1002/ppul.22766
PMCID: PMC3689612  PMID: 23401430
Wheeze; asthma; phenotype; lung function; cohort; atopy
3.  Prenatal alcohol exposure and childhood atopic disease: A Mendelian randomization approach☆ 
Background
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.
Objective
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.
Methods
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).
Results
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.
Conclusion
We have found no evidence to suggest that prenatal alcohol exposure increases the risk of asthma or atopy in childhood.
doi:10.1016/j.jaci.2013.04.051
PMCID: PMC3884122  PMID: 23806636
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
4.  Interaction of prenatal maternal smoking, interleukin 13 genetic variants and DNA methylation influencing airflow and airway reactivity 
Clinical Epigenetics  2013;5(1):22.
Background
Asthma is characterized by airflow limitation and airway reactivity (AR). Interleukin-13 (IL-13) is involved in the pathogenesis of asthma. Two functional SNPs, rs20541 and rs1800925, of the IL-13 gene (IL13) have been frequently associated with asthma-related lung functions. However, genetic variation alone does not fully explain asthma risk. DNA-methylation (DNA-M) is an epigenetic mechanism that regulates gene expression and can be influenced by both environment and genetic variants. To explore the interplay of prenatal maternal smoking, genetic variants and DNA-M, we used a two-stage model: (1) identifying cytosine phosphate guanine (CpG) sites where DNA-M is influenced by the interaction between genetic variants and maternal smoking during pregnancy (conditional methQTL (methylation quantitative trait loci)); and (2) determining the effect of the interaction between DNA-M of CpG (from stage 1) and SNPs (modifying genetic variants; modGV) on airflow limitation and AR in 245 female participants of the Isle of Wight birth cohort. DNA-M was assessed using the Illumina Infinium HumanMethylation450 BeadChip.
Findings
Six CpG sites were analyzed in stage 1. DNA-M at cg13566430 was influenced by interaction of maternal smoking during pregnancy and rs20541. In stage 2, genotype at rs1800925 interacted with DNA-M at cg13566430 significantly affecting airflow limitation (P = 0.042) and AR (P = 0.01).
Conclusion
Both genetic variants and environment affect DNA-M. This study supports the proposed two-stage model (methQTL and modGV) to study genetic variants, environment and DNA-M interactions in asthma-related lung function.
doi:10.1186/1868-7083-5-22
PMCID: PMC3892084  PMID: 24314122
Asthma genetics and epigenetics; Airway reactivity; DNA methylation; IL13 gene; Lung functions; Maternal smoking during pregnancy
5.  Dysregulation of Complement System and CD4+ T Cell Activation Pathways Implicated in Allergic Response 
PLoS ONE  2013;8(10):e74821.
Allergy is a complex disease that is likely to involve dysregulated CD4+ T cell activation. Here we propose a novel methodology to gain insight into how coordinated behaviour emerges between disease-dysregulated pathways in response to pathophysiological stimuli. Using peripheral blood mononuclear cells of allergic rhinitis patients and controls cultured with and without pollen allergens, we integrate CD4+ T cell gene expression from microarray data and genetic markers of allergic sensitisation from GWAS data at the pathway level using enrichment analysis; implicating the complement system in both cellular and systemic response to pollen allergens. We delineate a novel disease network linking T cell activation to the complement system that is significantly enriched for genes exhibiting correlated gene expression and protein-protein interactions, suggesting a tight biological coordination that is dysregulated in the disease state in response to pollen allergen but not to diluent. This novel disease network has high predictive power for the gene and protein expression of the Th2 cytokine profile (IL-4, IL-5, IL-10, IL-13) and of the Th2 master regulator (GATA3), suggesting its involvement in the early stages of CD4+ T cell differentiation. Dissection of the complement system gene expression identifies 7 genes specifically associated with atopic response to pollen, including C1QR1, CFD, CFP, ITGB2, ITGAX and confirms the role of C3AR1 and C5AR1. Two of these genes (ITGB2 and C3AR1) are also implicated in the network linking complement system to T cell activation, which comprises 6 differentially expressed genes. C3AR1 is also significantly associated with allergic sensitisation in GWAS data.
doi:10.1371/journal.pone.0074821
PMCID: PMC3792967  PMID: 24116013
6.  Genetic susceptibility to lung cancer and co-morbidities 
Journal of Thoracic Disease  2013;5(Suppl 5):S454-S462.
Lung cancer is a leading cause of cancer death and disease burden in many countries. Understanding of the biological pathways involved in lung cancer aetiology is required to identify key biomolecules that could be of significant clinical value, either as predictive, prognostic or diagnostic markers, or as targets for the development of novel therapies to treat this disease, in addition to smoking avoidance strategies. Genome-wide association studies (GWAS) have enabled significant progress in the past 5 years in investigating genetic susceptibility to lung cancer. Large scale, multi-cohort GWAS of mainly Caucasian, smoking, populations have identified strong associations for lung cancer mapped to chromosomal regions 15q [nicotinic acetylcholine receptor (nAChR) subunits: CHRNA3, CHRNA5], 5p (TERT-CLPTM1L locus) and 6p (BAT3-MSH5). Some studies in Asian populations of smokers have found similar risk loci, whereas GWAS in never smoking Asian females have identified associations in other chromosomal regions, e.g., 3q (TP63), that are distinct from smoking-related lung cancer risk loci. GWAS of smoking behaviour have identified risk loci for smoking quantity at 15q (similar genes to lung cancer susceptibility: CHRNA3, CHRNA5) and 19q (CYP2A6). Other genes have been mapped for smoking initiation and smoking cessation. In chronic obstructive pulmonary disease (COPD), which is a known risk factor for lung cancer, GWAS in large cohorts have also found CHRNA3 and CHRNA5 single nucleotide polymorphisms (SNPs) mapping at 15q as risk loci, as well as other regions at 4q31 (HHIP), 4q24 (FAM13A) and 5q (HTR4). The overlap in risk loci between lung cancer, smoking behaviour and COPD may be due to the effects of nicotine addiction; however, more work needs to be undertaken to explore the potential direct effects of nicotine and its metabolites in gene-environment interaction in these phenotypes. Goals of future genetic susceptibility studies of lung cancer should focus on refining the strongest risk loci in a wide range of populations with lung cancer, and integrating other clinical and biomarker information, in order to achieve the aim of personalised therapy for lung cancer.
doi:10.3978/j.issn.2072-1439.2013.08.06
PMCID: PMC3804872  PMID: 24163739
Lung cancer; genetics; pulmonary disease; chronic obstructive; genome-wide association study (GWAS)
7.  MUC5AC and inflammatory mediators associated with respiratory outcomes in the British 1946 birth cohort 
Respirology (Carlton, Vic.)  2013;18(6):1003-1010.
Background and objective: Dysregulation of respiratory mucins, MUC5AC in particular, has been implicated in respiratory disease and MUC5AC expression is up-regulated in response to environmental challenges and inflammatory mediators. The aim of this study was to examine the effect of genetic variation on susceptibility to common respiratory conditions.
Methods: The association of MUC5AC and the closely linked genes MUC2 and MUC5B with respiratory outcomes was tested in the MRC National Survey of Health and Development, a longitudinal birth cohort of men and women born in 1946. Also examined were the functional variants of the genes encoding inflammatory mediators, IL13, IL1B, IL1RN, TNFA and ERBB1, for which there is a likely influence on MUC5AC expression and were explored potential gene-gene interactions with these inflammatory mediators.
Results: Statistically significant associations between the 3'ter MUC5AC simple nucleotide polymorphism (SNP) rs1132440 and various non-independent respiratory outcomes (bronchitis, wheeze, asthma, hay fever) were reported while the adjacent loci show slight (but largely non-statistically significant) differences, presumably reflective of linkage disequilibrium (allelic association) across the region. A novel association between bronchitis and a non-synonymous functional ERBB1 SNP, rs2227983 (aka epidermal growth factor receptor:R497K, R521K) is also reported and evidence presented of interaction between MUC5AC and ERBB1 and between MUC5AC and IL1RN with respect to bronchitis. The ERBB1 result suggests a clear mechanism for a biological interaction in which the allelic variants of epidermal growth factor receptor differentially affect mucin expression.
Conclusions: The MUC5AC association and the interactions with inflammatory mediators suggest that genetically determined differences in MUC5AC expression alter susceptibility to respiratory disease.
SUMMARY AT A GLANCE
This longitudinal cohort study shows occurrence of the common respiratory conditions bronchitis, wheeze, asthma and hay fever to be associated with genetic variation in a mucin gene, MUC5AC. Functional variation in the epidermal growth factor receptor (epidermal growth factor receptor encoded by ERBB1) is also associated with bronchitis and modulates the MUC5AC effect.
doi:10.1111/resp.12092
PMCID: PMC3784974  PMID: 23551418
airway epithelium; asthma; genetics; inflammation; respiratory function test
8.  Defining the contribution of SNPs identified in asthma GWAS to clinical variables in asthmatic children 
BMC Medical Genetics  2013;14:100.
Background
Asthma genome-wide association studies (GWAS) have identified several asthma susceptibility genes with confidence; however the relative contribution of these genetic variants or single nucleotide polymorphisms (SNPs) to clinical endpoints (as opposed to disease diagnosis) remains largely unknown. Thus the aim of this study was to firstly bridge this gap in knowledge and secondly investigate whether these SNPs or those that are in linkage disequilibrium are likely to be functional candidates with respect to regulation of gene expression, using reported data from the ENCODE project.
Methods
Eleven of the key SNPs identified in eight loci from recent asthma GWAS were evaluated for association with asthma and clinical outcomes, including percent predicted FEV1, bronchial hyperresponsiveness (BHR) to methacholine, severity defined by British Thoracic Society steps and positive response to skin prick test, using the family based association test additive model in a well characterised UK cohort consisting of 370 families with at least two asthmatic children.
Results
GSDMB SNP rs2305480 (Ser311Pro) was associated with asthma diagnosis (p = 8.9×10-4), BHR (p = 8.2×10-4) and severity (p = 1.5×10-4) with supporting evidence from a second GSDMB SNP rs11078927 (intronic). SNPs evaluated in IL33, IL18R1, IL1RL1, SMAD3, IL2RB, PDE4D, CRB1 and RAD50 did not show association with any phenotype tested when corrected for multiple testing. Analysis using ENCODE data provides further insight into the functional relevance of these SNPs.
Conclusions
Our results provide further support for the role of GSDMB SNPs in determining multiple asthma related phenotypes in childhood asthma including associations with lung function and disease severity.
doi:10.1186/1471-2350-14-100
PMCID: PMC3849932  PMID: 24066901
Asthma; ENCODE; eQTL; GWAS; Clinical endpoints
9.  Effect of GSTM2-5 polymorphisms in relation to tobacco smoke exposures on lung function growth: a birth cohort study 
Background
Genetic variation within GSTM2-5 genes may interfere with detoxification of environmental compounds, thereby having a detrimental effect on lung function following exposures such as tobacco smoke. We aim to investigate the influence of variants and associated methylation in the GSTM gene cluster with changes in lung function growth during adolescence.
Methods
Growth in forced expiratory volume (FEV1), forced vital capacity (FVC), and change in FEV1/FVC ratio measures were obtained from children in the Isle of Wight birth cohort at ages 10 and 18. Illumina GoldenGate assays were used to genotype 10 tagging polymorphisms from GSTM2 (rs574344 and rs12024479), GSTM3 (rs1537236, rs7483, and rs10735234), GSTM4 (rs668413, rs560018, and rs506008), and GSTM5 (rs929166 and rs11807) genes. Diplotypes were generated in the software Phase 3.0.2. DNA methylation was measured in over 450,000 CpG sites using the Infinium HumanMethylation450 BeadChip (Illumina 450K) in a subsample of 245 18-year olds from the Isle of Wight birth cohort. Gender, age, in utero smoke exposure, secondhand smoke exposure (SHS), and current smoking status were assessed via questionnaire; smoke exposures were validated with urine cotinine. We used linear mixed models to estimate the effect of GSTM diplotypes on lung function across time and examine interactions with tobacco smoke.
Results
1,121 (77%) out of 1,456 children had information on lung function at ages 10 or 18. After adjustment for false discovery rate, one diplotype in GSTM3 had a detrimental effect on changes in FEV1 (p=0.03), and another diplotype in GSTM3 reduced FVC (p=0.02) over time. No significant interactions with smoking were identified. SHS significantly modified the relationship between diplotypes and methylation levels in one GSTM2 CpG site; however, this site did not predict lung function outcomes at age 18. Joint effects of GSTM loci and CpG sites located within these loci on adolescent lung growth were detected.
Conclusions
Diplotypes within GSTM2-5 genes are associated with lung function growth across adolescence, but do not appear to modify the effect of tobacco smoke exposures on adolescent lung growth. Interactions between DNA methylation and diplotypes should be taken into account to gain further understanding on lung function in adolescence.
doi:10.1186/1471-2466-13-56
PMCID: PMC3846453  PMID: 24004509
Smoking; Lung function; Diplotype; Human; Longitudinal study; Epigenetics; Methylation quantitative trait loci
11.  The effect of parental allergy on childhood allergic diseases depends on the sex of the child 
Background
Parent of origin effect is important in understanding the genetic basis of childhood allergic diseases and to improve our ability to identify high risk children.
Objective
To investigate parent of origin effect in childhood allergic diseases.
Methods
The Isle of Wight Birth Cohort (n=1,456) has been examined at 1, 2, 4, 10 and 18-years. Information on prevalence of asthma, eczema, rhinitis and environmental factors was obtained using validated questionnaires. Skin prick tests were carried out at ages 4, 10 and 18 year, and total IgE at 10 and 18 years. Parental history of allergic disease was assessed soon after the birth of the child when maternal IgE was also measured. Prevalence ratios (PR) and their 95% confidence intervals (CI) were estimated, applying log-linear models, adjusted for confounding variables.
Results
When stratified for sex of the child, maternal asthma was associated with asthma in girls [PR:1.91 (CI:1.34–2.72), p=0.0003], but not in boys [PR:1.29 (CI:0.85–1.96), p=0.23), while paternal asthma was associated with asthma in boys [PR:1.99 (CI:1.42–2.79), p<0.0001], but not in girls [PR: 1.03 (0.59–1.80) p=0.92). Maternal eczema increased the risk of eczema in girls [PR: 1.92 (CI: 1.37–2.68); p=0.0001] only, while paternal eczema did the same for boys (PR: 2.07 (CI:1.32–3.25); P=0.002). Similar trends were observed when the effect of maternal and paternal allergic disease was assessed for childhood atopy and when maternal total IgE was related to total IgE in children at age 10 and 18 years.
Conclusions
The current study indicates a sex dependent association of parental allergic conditions with childhood allergies; maternal allergy increasing the risk in girls and paternal allergy in boys. This has implications for childhood allergy prediction and prevention.
doi:10.1016/j.jaci.2012.03.042
PMCID: PMC3409323  PMID: 22607991
maternal; paternal; sex; cohort; parent of origin; atopy; asthma; eczema; rhinitis; allergy; IgE
12.  The methylation of the LEPR/LEPROT genotype at the promoter and body regions influence concentrations of leptin in girls and BMI at age 18 years if their mother smoked during pregnancy 
To determine whether DNA methylation (DNA-M) of the leptin receptor genotype (LEPR/LEPROT) links gestational smoking and leptin serum levels and BMI later in life, we focused on female offspring, 18 years of age, from the Isle of Wight Birth Cohort (IOWBC). Leptin binds to the leptin receptor encoded by the LEPR/LEPROT genotype. Using general linear models, we tested a two-stage model. First, we investigated whether single nucleotide polymorphisms (SNPs) acting as methylation quantitative trait loci (methQTLs) depending on gestational smoking were related to differentially methylated cytosine-phosphate-guanine (CpG) sites. In stage 2, we tested whether the selected CpG sites, in interaction with other SNPs (modifiable genetic variants, modGV), are associated with serum leptin and BMI (stage 2). Children from the IOWBC were followed from birth to age 18. Information on gestational smoking was gathered upon delivery. SNPs tagging LEPR and LEPROT genes were genotyped. Data on LEPR/LEPROT DNA-M and leptin were obtained from blood samples drawn at age 18; to determine BMI, height and weight were ascertained. Blood samples were provided by 238 girls. Of the 21 CpG sites, interactions between gestational smoking and SNPs were detected for 16 CpGs. Methylation of seven of the 16 CpGs were, in interaction with modGVs, associated with leptin levels at age 18 years. Two CpGs survived a multiple testing penalty and were also associated with BMI. This two-stage model may explain why maternal smoking has a long-term effect on leptin levels and BMI in girls at age 18 years.
PMCID: PMC3709113  PMID: 23875062
LEPR; LEPROT; leptin; CpG sites; in utero smoking exposure; rs12059300; BMI
13.  The interaction of genetic variants and DNA methylation of the interleukin-4 receptor gene increase the risk of asthma at age 18 years 
Clinical Epigenetics  2013;5(1):1.
Background
The occurrence of asthma is weakly explained by known genetic variants. Epigenetic marks, DNA methylation (DNA-M) in particular, are considered to add to the explanation of asthma. However, no etiological model has yet been developed that integrates genetic variants and DNA-M. To explore a new model, we focused on one asthma candidate gene, the IL-4 receptor (IL4R). We hypothesized that genetic variants of IL4R in interaction with DNA-M at cytosine-phosphate-guanine (CpG) sites jointly alter the risk of asthma during adolescence. Blood samples were collected at age 18 years from 245 female cohort participants randomly selected for methylation analysis from a birth cohort (n = 1,456, Isle of Wight, UK). Genome-wide DNA-M was assessed using the Illumina Infinium HumanMethylation450 BeadChip.
Results
Thirteen single nucleotide polymorphisms (SNPs) and twelve CpG sites of IL4R gene were analyzed. Based on linkage disequilibrium and association with asthma, eight SNPs and one CpG site were selected for further analyses. Of the twelve CpG sites in the IL4R gene, only methylation levels of cg09791102 showed an association with asthma at age 18 years (Wilcoxon test: P = 0.01). Log-linear models were used to estimate risk ratios (RRs) for asthma adjusting for uncorrelated SNPs within the IL4R gene and covariates. Testing for interaction between the eight SNPs and the methylation levels of cg09791102 on the risk for asthma at age 18 years, we identified the statistically significant interaction term of SNP rs3024685 × methylation levels of cg09791102 (P = 0.002; after adjusting for false discovery rate). A total of 84 participants had methylation levels ≤0.88, 112 participants between 0.89 and 0.90, and 35 between 0.91 and 0.92. For the SNP rs3024685 (‘CC’ vs. ‘TT’) at methylation levels of ≤0.85, 0.86, 0.90, 0.91, and 0.92, the RRs were 0.01, 0.04, 4.65, 14.76, 14.90, respectively (interaction effect, P = 0.0003).
Conclusions
Adjusting for multiple testing, our results suggest that DNA-M modulates the risk of asthma related to genetic variants in the IL4R gene. The strong interaction of one SNP and DNA-M is encouraging and provides a novel model of how a joint effect of genetic variants and DNA-M can explain occurrence of asthma.
doi:10.1186/1868-7083-5-1
PMCID: PMC3544634  PMID: 23286427
Interleukin-4 receptor gene; DNA methylation; Genetic variants; Asthma; Epigenetics
14.  Leukotriene B4 receptor locus gene characterisation and association studies in asthma 
BMC Medical Genetics  2012;13:110.
Background
Polymorphisms spanning genes involved in the production of leukotriene B4 (LTB4) e.g. ALOX5AP and LTA4H are associated with asthma susceptibility, suggesting a role for LTB4 in disease. The contribution of LTB4receptor polymorphism is currently unknown. The aim of this study was to characterise the genes for the two pivotal LTB4 receptors, LTB4R1 and LTB4R2 in lung tissue and determine if polymorphisms spanning these genes are associated with asthma and disease severity.
Methods
Rapid amplification of cDNA ends (RACE) was used to characterise the LTB4R1 and LTB4R2 gene structure in lung. The LTB4R1/2 locus on chromosome 14q11.2 was screened for polymorphic variation. Six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in 370 Caucasian asthma families and 299 Adult Asthma Individuals (n=1877 total) and were evaluated for association with asthma and severity (BTS) outcome measures using Family Based Association Test, linear regression and chi square.
Results
LTB4R1 has complex mRNA arrangement including multiple 5′-untranslated exons, suggesting additional levels of regulation. Three potential promoter regions across the LTB4R1/2 locus were identified with some airway cell specificity. 22 SNPs (MAF>0.01) were validated across the LTB4R locus in the Caucasian population. LTB4R1 and LTB4R2 SNPs were not associated with asthma susceptibility, FEV1 or severity.
Conclusions
LTB4R1 and LTB4R2 shows splice variation in the 5′-untranslated region and multiple promoter regions. The functional significance of this is yet to be determined. Both receptor genes were shown to be polymorphic. LTB4R polymorphisms do not appear to be susceptibility markers for the development of asthma in Caucasian subjects.
doi:10.1186/1471-2350-13-110
PMCID: PMC3607986  PMID: 23167751
Association; Asthma; Family based association test; Leukotriene; Leukotriene B4 receptor; RACE; Severity
15.  Identification of ATPAF1 as a novel candidate gene for asthma in children 
Background
Asthma is a common disease of children with a complex genetic origin. Understanding the genetic basis of asthma susceptibility will allow disease prediction and risk stratification.
Objective
We sought to identify asthma susceptibility genes in children.
Methods
A nested case-control genetic association study of children of Caucasian European ancestry from a birth cohort was conducted. Single nucleotide polymorphisms (SNPs, n=116,024) were genotyped in pools of DNA samples from cohort children with physician-diagnosed asthma (n=112) and normal controls (n=165). A genomic region containing the ATPAF1 gene was significantly associated with asthma. Additional SNPs within this region were genotyped in individual samples from the same children and in eight independent study populations consisting of Caucasian, African American, Hispanic, or other ancestries. SNPs were also genotyped or imputed in two consortia control populations. ATPAF1 expression was measured in bronchial biopsies from asthmatics and controls.
Results
Asthma was associated with a cluster of SNPs and SNP haplotypes containing the ATPAF1 gene with two SNPs achieving significance at a genome-wide level (p=2.26×10−5 to 2.2×10−8). Asthma severity was also associated with SNPs and haplotypes in the primary population. SNP and/or gene-level associations were confirmed in the four non-Hispanic populations. Haplotype associations were confirmed in the non-Hispanic populations (p=0.045 to 0.0009). ATPAF1 total RNA expression was significantly (p<0.01) higher in bronchial biopsies from asthmatics than controls.
Conclusion
Genetic variation in the ATPAF1 gene predisposes children of different ancestry to asthma.
doi:10.1016/j.jaci.2011.04.058
PMCID: PMC3185108  PMID: 21696813
asthma; ATPAF1; children; gene; genetic; genome-wide association; purinergic; respiratory; single nucleotide polymorphism; SNP
16.  Interleukin-13, but Not Indomethacin, Increases Cysteinyl-Leukotriene Synthesis in Human Lung Macrophages 
Journal of Allergy  2011;2012:348741.
Aspirin-exacerbated respiratory disease (AERD) is associated with constitutively elevated synthesis of bronchoconstrictor cysteinyl-leukotrienes, associated with increased expression of leukotriene (LT)C4 synthase and Th2 cytokines and airway eosinophilia. We examined whether interleukin-13 can increase LTC4 synthase gene transcription and cysteinyl-leukotriene synthesis in macrophages isolated from resected human lung tissue and whether an NSAID (indomethacin) can trigger further cysteinyl-leukotriene synthesis in these cells. Overnight culture of human lung macrophages with IL-13 (10 ng/mL) increased spontaneous and ionophore-stimulated production of cysteinyl-leukotrienes by 42% (P = 0.02) and 52% (P = 0.005), respectively, as quantified by enzyme immunoassays, but PCR gene transcription assays did not demonstrate an effect on LTC4S mRNA. The addition of indomethacin (100 μM) did not modulate cysteinyl-leukotriene production in either IL-13-treated or untreated macrophages. We conclude that while IL-13 enhances cysteinyl-leukotriene synthesis in human lung macrophages, it does not replicate the enhanced LTC4 synthase expression observed in the AERD lung nor confer sensitivity to NSAIDs.
doi:10.1155/2012/348741
PMCID: PMC3205618  PMID: 22121385
17.  Adjusting wheal size measures to correct atopy misclassification 
Purpose:
Skin prick testing (SPT) is fundamental to the practice of clinical allergy identifying relevant allergens and predicting the clinical expression of disease. Wheal sizes on SPT are used to identify atopic cases, and the cut-off value for a positive test is commonly set at 3 mm. However, the measured wheal sizes do not solely reflect the magnitude of skin reaction to allergens, but also skin reactivity (reflected in the size of histamine reaction) and other random or non-random factors. We sought to estimate wheal sizes exclusively due to skin response to allergens and propose gender-specific cutoff points of atopy.
Methods:
We developed a Bayesian method to adjust observed wheal sizes by excluding histamine and other factor effects, based on which revised cutoff points are proposed for males and females, respectively. The method is then applied to and intensively evaluated using a study population aged 18, at a location on the Isle of Wight in the United Kingdom. To evaluate the proposed approach, two sample t-tests for population means and proportion tests are applied.
Results:
Four common aeroallergens, house dust mite (HDM), grass pollen, dog dander, and alternaria are considered in the study. Based on 3 mm cutoff, males tend to be more atopic than females (P-values are between 0.00087 and 0.062). After applying the proposed methods to adjust wheal sizes, our findings suggest that misclassifications of atopy occur more often in males. Revised allergen-specific cutoff values are proposed for each gender.
Conclusion:
To reduce the gender discrepancy, we may have two potentially convenient solutions. One way is to apply allergen-specific and gender-specific cutoff values following the proposed method. Alternatively, we can revise the concentration of allergens in the SPT solutions but keep the cutoff values unchanged, which may be more convenient to clinicians.
doi:10.2147/IJGM.S22193
PMCID: PMC3160870  PMID: 21887114
SPT; atopy; Bayesian method; joint modeling; misclassification
18.  Glutathione-S-transferase genes and asthma phenotypes: a Human Genome Epidemiology (HuGE) systematic review and meta-analysis including unpublished data 
Background Oxidative stress is thought to be involved in the pathogenesis of asthma. Glutathione-S-transferase (GST) enzymes, which play an important role in antioxidant defences, may therefore influence asthma risk. Two common deletion polymorphisms of GSTM1 and GSTT1 genes and the GSTP1 Ile105Val polymorphism have been associated with asthma in children and adults, but results are inconsistent across studies.
Methods Systematic review and meta-analysis of the effects of GST genes on asthma, wheezing and bronchial hyper-responsiveness (BHR), with inclusion of unpublished data from three studies, including the large Avon Longitudinal Study of Parents and Children (ALSPAC). Random effect or fixed effect models were used as appropriate, and sensitivity analyses were performed to assess the impact of study characteristics and quality on pooled results.
Results The meta-analyses of GSTM1 (n = 22 studies) and GSTT1 (n = 19) showed increased asthma risk associated with the null genotype, but there was extreme between-study heterogeneity and publication bias and the association disappeared when meta-analysis was restricted to the largest studies. Meta-analysis of GSTP1 Ile105Val (n = 17) and asthma suggested a possible protective effect of the Val allele, but heterogeneity was extreme. Few studies evaluated wheezing and BHR and most reported no associations, although weak evidence was found for positive associations of GSTM1 null and GSTP1 Val allele with wheezing and a negative association of GSTP1 Val allele with BHR.
Conclusions Our findings do not support a substantial role of GST genes alone in the development of asthma. Future studies of large size should focus on interactions of GST genes with environmental oxidative exposures and with other genes involved in antioxidant pathways. Quality of study conduct and reporting needs to be improved to increase credibility of the evidence accumulating over time.
doi:10.1093/ije/dyp337
PMCID: PMC2846443  PMID: 20032267
Meta-analysis; systematic review; glutathione-S-transferase genes; GSTM1 gene; GSTT1 gene; GSTP1 gene; asthma; wheezing; bronchial responsiveness; The Avon Longitudinal Study of Parents and Children (ALSPAC)
19.  Effect of β2-Adrenergic Receptor Polymorphism in Asthma Control of Patients Receiving Combination Treatment 
Yonsei Medical Journal  2009;50(2):182-188.
Purpose
Combination treatment of inhaled corticosteroid (ICS) plus long-acting β2-agonist (LABA) is widely used as a maintenance regimen for the management of asthma. This study evaluated the effect of the β2-adrenergic receptor (ADRB2) polymorphism on lung function and asthma control with regular use of combination treatment of an inhaled ICS plus LABA.
Materials and Methods
43 Korean asthmatics who were symptomatic despite regular ICS use for at least 3 months were enrolled. For a 2-week run-in period, they received ICS (budesonide 800 µg/day) plus terbutaline (5 µg prn). as needed. During the 24-week active treatment period, they received budesonide 160 µg and formoterol 4.5 µg b.i.d. as maintenance and rescue medication. Pulmonary function and quality of life scores were monitored every 8 weeks; morning/evening peak expiratory flow meter (PEFR) was recorded daily. Patients were genotyped for ADRB2 Arg16Gly using single base extension methodology.
Results
During the run-in period, there were no significant between-group differences in lung function; after 8 weeks of active treatment, Arg/Arg patients had significantly higher forced expiratory volume in 1 secord (FEV1) and maximal mid-expiratory flow (MMEF) (p = 0.023 and p = 0.021, respectively), and better asthma control and quality of life after 24 weeks (p = 0.016 and p = 0.028, respectively). During treatment, there was a greater improvement in morning/evening PEFR in Arg/Arg patients.
Conclusion
Asthmatic patients with the Arg/Arg genotype at codon 16 of ADRB2 achieve better asthma control with long-term regular use of combined budesonide and formoterol treatment, suggesting that the ADRB2 genotype may dictate choice of treatment strategy.
doi:10.3349/ymj.2009.50.2.182
PMCID: PMC2678690  PMID: 19430548
β2-adrenergic receptor polymorphism; long-acting β2-agonist; bronchodilating effect
20.  Infection of Mice with Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) Suppresses Allergen-induced Airway Eosinophilia  
It has been proposed that the increase in prevalence and severity of atopic disorders inversely correlates with exposure to infectious diseases such as tuberculosis. We have investigated this issue by combining an intranasal Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) infection with a murine model of allergen, (ovalbumin [OVA]) induced airway eosinophilia. BCG infection either 4 or 12 wk before allergen airway challenge resulted in a 90–95 and 60–70% reduction in eosinophilia within the lungs, respectively, compared to uninfected controls. The inhibition of airway eosinophilia correlated with a reduced level of IL-5 production by T cells from the lymph node draining the site of OVA challenge. Interestingly, BCG infection of the lung had no effect on IgG1 and IgE OVA-specific serum immunoglobulin or blood eosinophil levels. Furthermore, BCG-induced inhibition of airway eosinophilia was strongly reduced in interferon (IFN)-γ receptor–deficient mice and could be partially reversed by intranasal IL-5 application. Intranasal BCG infections could also reduce the degree of lung eosinophilia and IL-5 produced by T cells after Nippostrongylus brasiliensis infection. Taken together, our data suggest that IFN-γ produced during the T helper cell (Th)1 immune response against BCG suppresses the development of local inflammatory Th2 responses in the lung. Most importantly, this inhibition did not extend to the systemic immunoglobulin response against OVA. Our data support the view that mycobacterial infections have the potential to suppress the development of atopic disorders in humans.
PMCID: PMC2212158  PMID: 9463406
21.  Interactive effect of STAT6 and IL13 gene polymorphisms on eczema status: results from a longitudinal and a cross-sectional study 
BMC Medical Genetics  2013;14:67.
Background
Eczema is a prevalent skin disease that is mainly characterized by systemic deviation of immune response and defective epidermal barrier. Th2 cytokines, such as IL-13 and transcription factor STAT6 are key elements in the inflammatory response that characterize allergic disorders, including eczema. Previous genetic association studies showed inconsistent results for the association of single nucleotide polymorphisms (SNPs) with eczema. Our aim was to investigate whether SNPs in IL13 and STAT6 genes, which share a biological pathway, have an interactive effect on eczema risk.
Methods
Data from two independent population-based studies were analyzed, namely the Isle of Wight birth cohort study (IOW; n = 1,456) and for the purpose of replication the Swansea PAPA (Poblogaeth Asthma Prifysgol Abertawe; n = 1,445) cross-sectional study. Log-binomial regressions were applied to (i) account for the interaction between IL13 (rs20541) and STAT6 (rs1059513) polymorphisms and (ii) estimate the combined effect, in terms of risk ratios (RRs), of both risk factors on the risk of eczema.
Results
Under a dominant genetic model, the interaction term [IL13 (rs20541) × STAT6 (rs1059513)] was statistically significant in both studies (IOW: adjusted Pinteraction = 0.046; PAPA: Pinteraction = 0.037). The assessment of the combined effect associated with having risk genotypes in both SNPs yielded a 1.52-fold increased risk of eczema in the IOW study (95% confidence interval (CI): 1.05 – 2.20; P = 0.028) and a 2.01-fold higher risk of eczema (95% CI: 1.29 – 3.12; P = 0.002) in the PAPA study population.
Conclusions
Our study adds to the current knowledge of genetic susceptibility by demonstrating for the first time an interactive effect between SNPs in IL13 (rs20541) and STAT6 (rs1059513) on the occurrence of eczema in two independent samples. Findings of this report further support the emerging evidence that points toward the existence of genetic effects that occur via complex networks involving gene-gene interactions (epistasis).
doi:10.1186/1471-2350-14-67
PMCID: PMC3700873  PMID: 23815671
Eczema; Gene-gene Interaction; Epistasis; STAT6; IL13; Genetic Association Study
22.  MUC5AC and inflammatory mediators associated with respiratory outcomes in the British 1946 birth cohort 
Respirology (Carlton, Vic.)  2013;18(6):1003-1010.
Background and objective: Dysregulation of respiratory mucins, MUC5AC in particular, has been implicated in respiratory disease and MUC5AC expression is up-regulated in response to environmental challenges and inflammatory mediators. The aim of this study was to examine the effect of genetic variation on susceptibility to common respiratory conditions.
Methods: The association of MUC5AC and the closely linked genes MUC2 and MUC5B with respiratory outcomes was tested in the MRC National Survey of Health and Development, a longitudinal birth cohort of men and women born in 1946. Also examined were the functional variants of the genes encoding inflammatory mediators, IL13, IL1B, IL1RN, TNFA and ERBB1, for which there is a likely influence on MUC5AC expression and were explored potential gene-gene interactions with these inflammatory mediators.
Results: Statistically significant associations between the 3'ter MUC5AC simple nucleotide polymorphism (SNP) rs1132440 and various non-independent respiratory outcomes (bronchitis, wheeze, asthma, hay fever) were reported while the adjacent loci show slight (but largely non-statistically significant) differences, presumably reflective of linkage disequilibrium (allelic association) across the region. A novel association between bronchitis and a non-synonymous functional ERBB1 SNP, rs2227983 (aka epidermal growth factor receptor:R497K, R521K) is also reported and evidence presented of interaction between MUC5AC and ERBB1 and between MUC5AC and IL1RN with respect to bronchitis. The ERBB1 result suggests a clear mechanism for a biological interaction in which the allelic variants of epidermal growth factor receptor differentially affect mucin expression.
Conclusions: The MUC5AC association and the interactions with inflammatory mediators suggest that genetically determined differences in MUC5AC expression alter susceptibility to respiratory disease.
SUMMARY AT A GLANCE
This longitudinal cohort study shows occurrence of the common respiratory conditions bronchitis, wheeze, asthma and hay fever to be associated with genetic variation in a mucin gene, MUC5AC. Functional variation in the epidermal growth factor receptor (epidermal growth factor receptor encoded by ERBB1) is also associated with bronchitis and modulates the MUC5AC effect.
doi:10.1111/resp.12092
PMCID: PMC3786313
airway epithelium; asthma; genetics; inflammation; respiratory function test
23.  Next generation exome sequencing of paediatric inflammatory bowel disease patients identifies rare and novel variants in candidate genes 
Gut  2012;62(7):977-984.
Background
Multiple genes have been implicated by association studies in altering inflammatory bowel disease (IBD) predisposition. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition plays a more substantial role in this group.
Objective
To identify the spectrum of rare and novel variation in known IBD susceptibility genes using exome sequencing analysis in eight individual cases of childhood onset severe disease.
Design
DNA samples from the eight patients underwent targeted exome capture and sequencing. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, and identify and annotate variants where patient sequence differed from the reference sequence. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued.
Results
Across the panel of 169 known IBD susceptibility genes, approximately 300 variants in 104 genes were found. Excluding splicing and HLA-class variants, 58 variants across 39 of these genes were classified as rare, with an alternative allele frequency of <5%, of which 17 were novel. Only two patients with early onset Crohn's disease exhibited rare deleterious variations within NOD2: the previously described R702W variant was the sole NOD2 variant in one patient, while the second patient also carried the L1007 frameshift insertion. Both patients harboured other potentially damaging mutations in the GSDMB, ERAP2 and SEC16A genes. The two patients severely affected with ulcerative colitis exhibited a distinct profile: both carried potentially detrimental variation in the BACH2 and IL10 genes not seen in other patients.
Conclusion
For each of the eight individuals studied, all non-synonymous, truncating and frameshift mutations across all known IBD genes were identified. A unique profile of rare and potentially damaging variants was evident for each patient with this complex disease.
doi:10.1136/gutjnl-2011-301833
PMCID: PMC3686259  PMID: 22543157
IBD-genetics; inflammatory bowel disease; crohn's disease; paediatric gastroenterology; ulcerative colitis; zollinger ellison syndrome,
24.  Genome-wide DNA methylation analysis of patients with imprinting disorders identifies differentially methylated regions associated with novel candidate imprinted genes 
Journal of Medical Genetics  2014;51(4):229-238.
Background
Genomic imprinting is allelic restriction of gene expression potential depending on parent of origin, maintained by epigenetic mechanisms including parent of origin-specific DNA methylation. Among approximately 70 known imprinted genes are some causing disorders affecting growth, metabolism and cancer predisposition. Some imprinting disorder patients have hypomethylation of several imprinted loci (HIL) throughout the genome and may have atypically severe clinical features. Here we used array analysis in HIL patients to define patterns of aberrant methylation throughout the genome.
Design
We developed a novel informatic pipeline capable of small sample number analysis, and profiled 10 HIL patients with two clinical presentations (Beckwith–Wiedemann syndrome and neonatal diabetes) using the Illumina Infinium Human Methylation450 BeadChip array to identify candidate imprinted regions. We used robust statistical criteria to quantify DNA methylation.
Results
We detected hypomethylation at known imprinted loci, and 25 further candidate imprinted regions (nine shared between patient groups) including one in the Down syndrome critical region (WRB) and another previously associated with bipolar disorder (PPIEL). Targeted analysis of three candidate regions (NHP2L1, WRB and PPIEL) showed allelic expression, methylation patterns consistent with allelic maternal methylation and frequent hypomethylation among an additional cohort of HIL patients, including six with Silver–Russell syndrome presentations and one with pseudohypoparathyroidism 1B.
Conclusions
This study identified novel candidate imprinted genes, revealed remarkable epigenetic convergence among clinically divergent patients, and highlights the potential of epigenomic profiling to expand our understanding of the normal methylome and its disruption in human disease.
doi:10.1136/jmedgenet-2013-102116
PMCID: PMC3963529  PMID: 24501229
Epigenetics; Genome-wide; Imprinting
25.  Interplay of Filaggrin Loss-of-Function Variants, Allergic Sensitization, and Eczema in a Longitudinal Study Covering Infancy to 18 Years of Age 
PLoS ONE  2012;7(3):e32721.
Background
Immune specific genes as well as genes regulating the formation of skin barrier are major determinants for eczema manifestation. There is a debate as to whether allergic sensitization and filaggrin gene (FLG) variants lead to eczema or FLG variants and eczema increase the risk of allergic sensitization. To investigate the time-order between eczema and allergic sensitization with respect to FLG variants, data from a large prospective study covering infancy to late adolescence were analyzed.
Methodology/Principal Findings
Repeated measurements of 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). Three transition periods were analyzed: age 1-or-2 to 4, 4 to 10, and 10 to 18 years. FLG variants were genotyped in 1,150 participants. Over the three transition periods, in temporal sequence analyses of initially eczema-free participants, the combined effect of FLG variants and allergic sensitization showed a 2.92-fold (95% CI: 1.47–5.77) increased risk ratio (RR) of eczema in subsequent examinations. This overall risk was more pronounced at a younger age (transition period 1-or-2 to 4, RR = 6.47, 95% CI: 1.96–21.33). In contrast, FLG variants in combination with eczema showed a weaker, but significant, risk ratio for subsequent allergic sensitization only up to 10 years of age.
Conclusions/Significance
Taking the time order into account, this prospective study demonstrates for the first time, that a combination of FLG variants and allergic sensitization increased the risk of eczema in subsequent years. Also FLG variants interacted with eczema and increased the risk of subsequent allergic sensitization, which, was limited to the younger age. Hence, early restoration of defective skin barrier could prevent allergic sensitization and subsequently reduce the risk of eczema development.
doi:10.1371/journal.pone.0032721
PMCID: PMC3293849  PMID: 22403702

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