Blood transcriptomic signature in type-2 biomarker-low severe asthma and asthma control.

BACKGROUND: Patients with type-2 (T2) cytokine-low severe asthma often have persistent symptoms despite suppression of T2 inflammation with corticosteroids. OBJECTIVES: We sought to analyze whole blood transcriptome from 738 samples in T2-biomarker-high/-low patients with severe asthma to relate transcriptomic signatures to T2 biomarkers and asthma symptom scores. METHODS: Bulk RNA-seq data were generated for blood samples (baseline, week 24, week 48) from 301 participants recruited to a randomized clinical trial of corticosteroid optimization in severe asthma. Unsupervised clustering, differential gene expression analysis, and pathway analysis were performed. Patients were grouped by T2-biomarker status and symptoms. Associations between clinical characteristics and differentially expressed genes (DEGs) associated with biomarker and symptom levels were investigated. RESULTS: Unsupervised clustering identified 2 clusters; cluster 2 patients were blood eosinophil-low/symptom-high and more likely to be receiving oral corticosteroids (OCSs). Differential gene expression analysis of these clusters, with and without stratification for OCSs, identified 2960 and 4162 DEGs, respectively. Six hundred twenty-seven of 2960 genes remained after adjusting for OCSs by subtracting OCS signature genes. Pathway analysis identified dolichyl-diphosphooligosaccharide biosynthesis and assembly of RNA polymerase I complex as significantly enriched pathways. No stable DEGs were associated with high symptoms in T2-biomarker-low patients, but numerous associated with elevated T2 biomarkers, including 15 that were upregulated at all time points irrespective of symptom level. CONCLUSIONS: OCSs have a considerable effect on whole blood transcriptome. Differential gene expression analysis demonstrates a clear T2-biomarker transcriptomic signature, but no signature was found in association with T2-biomarker-low patients, including those with a high symptom burden.

Genome-wide association study to identify genetic determinants of severe asthma.

BACKGROUND: The genetic basis for developing asthma has been extensively studied. However, association studies to date have mostly focused on mild to moderate disease and genetic risk factors for severe asthma remain unclear. OBJECTIVE: To identify common genetic variants affecting susceptibility to severe asthma. METHODS: A genome-wide association study was undertaken in 933 European ancestry individuals with severe asthma based on Global Initiative for Asthma (GINA) criteria 3 or above and 3346 clean controls. After standard quality control measures, the association of 480 889 genotyped single nucleotide polymorphisms (SNPs) was tested. To improve the resolution of the association signals identified, non-genotyped SNPs were imputed in these regions using a dense reference panel of SNP genotypes from the 1000 Genomes Project. Then replication of SNPs of interest was undertaken in a further 231 cases and 1345 controls and a meta-analysis was performed to combine the results across studies. RESULTS: An association was confirmed in subjects with severe asthma of loci previously identified for association with mild to moderate asthma. The strongest evidence was seen for the ORMDL3/GSDMB locus on chromosome 17q12-21 (rs4794820, p=1.03×10((-8)) following meta-analysis) meeting genome-wide significance. Strong evidence was also found for the IL1RL1/IL18R1 locus on 2q12 (rs9807989, p=5.59×10((-8)) following meta-analysis) just below this threshold. No novel loci for susceptibility to severe asthma met strict criteria for genome-wide significance. CONCLUSIONS: The largest genome-wide association study of severe asthma to date was carried out and strong evidence found for the association of two previously identified asthma susceptibility loci in patients with severe disease. A number of novel regions with suggestive evidence were also identified warranting further study.

Association between exhaled breath condensate nitrate + nitrite levels with ambient coarse particle exposure in subjects with airways disease.

OBJECTIVES: Studies of individual inflammatory responses to exposure to air pollution are few but are important in defining the most sensitive markers in better understanding pathophysiological pathways in the lung. The goal of this study was to assess whether exposure to airborne particles is associated with oxidative stress in an epidemiological setting. METHODS: The authors assessed exposure to particulate matter air pollution in four European cities in relation to levels of nitrite plus nitrate (NOx) in exhaled breath condensate (EBC) measurements in 133 subjects with asthma or chronic obstructive pulmonary disease using an EBC capture method developed for field use. In each subject, three measurements were collected. Exposure measurements included particles smaller than 10 µm (PM(10)), smaller than 2.5 µm (PM(2.5)) and particle number counts at a central site, outdoors near the subject's home and indoors. RESULTS: There were positive and significant relationships between EBC NOx and coarse particles at the central sampling sites (increase of 20.4% (95% CI 6.1% to 36.6%) per 10 µg/m(3) increase of coarse particles of the previous day) but not between EBC NOx and other particle measures. Associations tended to be stronger in subjects not taking steroid medication. CONCLUSIONS: An association was found between exposure to ambient coarse particles at central sites and EBC NOx, a marker of oxidative stress. The lack of association between PM measures more indicative of personal exposures (particularly indoor exposure) means interpretation should be cautious. However, EBC NOx may prove to be a marker of PM-induced oxidative stress in epidemiological studies.

Non random usage of T cell receptor alpha gene expression in atopy using anchored PCR.

The T cell receptor (TCR) alpha beta heterodimer recognises antigenic peptide fragments presented by Class II MHC. This interaction initiates T cell activation and cytokine release with subsequent recruitment of inflammatory cells. Previous work from our group suggests a qualitative difference in variable alpha gene expression in atopy as compared to non atopic controls. In this study we examine TCR alpha repertoire using anchored PCR to provide a quantitative assessment of the V alpha and J alpha repertoire. One atopic (DRB1*0701,DRB1*15: DRB4*0101, DRB5*01: DQB1* 0303, DQB1*601/2) and one non-atopic (DRB1*0701,DRB1*03011/2: DRB4*01, DRB3*0x: DQB1* 0303, DQB1*0201/2) control were studied. Variable gene usage was markedly limited in the atopic individual. V alpha 1, 3, 8 accounted for 60% and J alpha 12, 31 30% of the gene usage. There was evidence of preferential V alpha-J alpha gene pairing and clonal expansion. We conclude that there is a marked non random TCR alpha gene distribution in atopy using both V alpha family and anchored PCR. This may be due in part to antigen driven clonal expansion.

A critical review of the use of Clara cell secretory protein (CC16) as a biomarker of acute or chronic pulmonary effects.

Biomarkers associated with asthma aetiology and exacerbation have been sought to shed light on this multifactorial disease. One candidate is the serum concentration of the Clara cell secretory protein (CC16, sometimes referred to as CC10 or uteroglobin). In this review, we examine serum CC16's relation to asthma aetiology and exacerbation. There is evidence that acute exposures to certain pulmonary irritants can cause a transient increase in serum CC16 levels, and limited evidence also suggests that a transient increase in serum CC16 levels can be caused by a localized pulmonary inflammation. Research also indicates that a transient increase in serum CC16 is not associated with measurable pulmonary damage or impairment of pulmonary function. The biological interpretation of chronic changes in serum CC16 is less clear. Changes in serum CC16 concentrations (either transient or chronic) are not specific to any one agent, disease state, or aetiology. This lack of specificity limits the use of serum CC16 as a biomarker of specific exposures. To date, many of the critical issues that must be understood before serum CC16 levels can have an application as a biomarker of effect or exposure have not been adequately addressed.

Association study of asthma and atopy traits and chromosome 5q cytokine cluster markers.

BACKGROUND: Linkage studies have provided evidence for the presence of gene(s) in the 5q cytokine cluster region which control total serum immunoglobulin E (IgE) concentration, and bronchial hyperreactivity (BHR). However, the identification of the gene(s) involved has been confounded by the lack of power of the published linkage studies and the presence of multiple candidate genes mapped to the region. OBJECTIVE: To define the important loci on 5q31-33 which are implicated in the control of total serum IgE and BHR through a case/control study of association. METHODS: We performed an association study between 11 polymorphic markers (spanning the region 5q31.1-33.1) and total serum IgE and BHR traits. A case/control sample of 181 individuals was drawn from a larger set of 2415 adults, sampled at random from a district in Nottingham, UK. Half of the subjects in this case/control sample were hyperreactive to methacholine and asthmatic (cases), while the other half were non-reactive and non-asthmatic (controls). Association analysis was performed using the non-parametric chi-squared and Mann-Whitney U-tests. RESULTS: We observed no evidence of strong allelic association between any of the above markers and the studied traits. Markers D5S404, interferon regulatory factor 1 (IRF-1) and D5S210 showed evidence of borderline association with BHR (P = 0.04, 0.03 and 0.04 respectively), and D5S404 showed borderline significance with IgE levels (P = 0.029). CONCLUSIONS: This study presents evidence against the presence of a strong association between markers mapped to 5q31-33 and either BHR or total serum IgE. The significance of the weaker associations observed with markers D4S404, IRF-1 and D5S210 is not clear. Whether this represents a type I error secondary to multiple hypothesis testing or a true association is uncertain.

An association study between the Clara cell secretory protein CC16 A38G polymorphism and asthma phenotypes.

BACKGROUND: Previously, an association has been reported between an increased risk of asthma and a polymorphism in the Clara cell secretory protein (CC16) gene [namely, an adenine to guanine substitution in the CC16 gene at position 38 (A38G) downstream from the transcription initiation site within the noncoding region of exon 1]. Homozygous individuals for the polymorphic sequence (AA genotype) were reported to have a significant (6.9 fold) increased risk of developing asthma. This finding has not been confirmed independently. OBJECTIVE: To validate the association of CC16 A38G polymorphism to asthma in a separate well-characterized population through a case-control study. METHODS: We conducted an association study using a sample of 217 unrelated Northern European Caucasians. Individuals were clinically characterized by a validated respiratory questionnaire, spirometry and bronchial reactivity measurement, and genotyped for the A38G polymorphism using PCR and restriction digestion. Association analysis was performed using the nonparametric Chi-squared tests. RESULTS: In the unselected population, 43.3% participants were homozygous for the CC16*G allele and 45.4% were heterozygous (AG). We observed no significant difference in the distribution of positive bronchial reactivity to methacholine (at FEV1 PC20 of

Linkage/association study of a locus modulating total serum IgE on chromosome 14q13-24 in families with asthma.

BACKGROUND: A study was undertaken to validate a locus modulating total serum IgE levels on 14q13-24. METHODS: A linkage and association study was performed between total serum IgE and a panel of seven microsatellites which map to the 14q13-24 region in 69 families with asthma recruited from Leeds, UK. RESULTS: Non-parametric, multipoint, sib pair analysis showed no evidence of genetic linkage between the quantitative trait "log IgE" and any of the tested markers. However, a significant association was observed between locus D14S63 (14q23) and total serum IgE (p = 0.017). Allelic analysis showed an association between low total IgE and allele 157 of D14S63 (p = 0.01, OR = 0.63, 95% CI 0.44 to 0.90). Modelling of allele 157 genotypes as a continuous covariate indicated evidence of a significant inverse linear trend across the three genotypes where 157 homozygotes had the lowest mean log IgE (p = 0.045). Association of D14S63 with log IgE was confirmed in the analysis of a combined dataset of 53 families from Southampton, UK and the 69 families from Leeds (total 122 families). An association was observed at the locus level (p = 0.022) and the allelic level where allele 165 showed an association with high total IgE (p = 0.001, OR = 3.79, 95% CI 1.54 to 9.7) and allele 157 showed an association with low total IgE (p = 0.041, OR = 0.77, 95% CI 0.6 to 0.99). The transmission disequilibrium test was positive for allele 165 (p<0.05) and negative for allele 157 (p>0.05). CONCLUSIONS: Despite the lack of linkage, the findings of this study support the previous observation of a gene(s) at 14q23 that modulates total serum IgE.

Lack of linkage between chromosome 5q23-33 markers and IgE/bronchial hyperreactivity in 67 Scottish families.

BACKGROUND: Raised serum immunoglobulin E (IgE) and bronchial hyperreactivity (BHR) are risk factors for the expression of the asthma phenotype. Previous studies have reported evidence for linkage between these traits and markers on the 5q23-33 cytokine gene cluster. OBJECTIVE: To test for linkage between total serum IgE/BHR and microsatellite markers which map to the 5q23-33 region in an ethnically distinct cohort of families from Aberdeen, Scotland. METHODS: We performed a linkage study between five polymorphic markers (spanning the chromosome 5q23-33 region) and total serum IgE and BHR traits. A cohort of 67 families, who were recruited originally to study the natural history of wheeze, were clinically characterized and genotyped for D5S404, IL4, IRF-1, IL9, D5S436 markers. Linkage analyses were performed using the nonparametric Haseman-Elston algorithm for the quantitative trait log IgE, and the nonparametric LOD score (NPL-score) of the GENEHUNTER package for the qualitative traits serum IgE and BHR. RESULTS: The results of the nonparametric linkage analysis using either the Haseman-Elston algorithm or NPL-score were consistent and showed no evidence for linkage with IgE. There was also no evidence for linkage between the BHR traits (at cut-off values of PD20FEV1 < 8 mmol and 16 mmol) and any of the tested five microsatellite markers. CONCLUSIONS: This study presents evidence against the presence of a gene with a major effect on total serum IgE or BHR in the 5q23-33 region, in this ethnic group.

Suggestive evidence for genetic linkage between IgE phenotypes and chromosome 14q markers.

Chromosome 14q was screened for loci modulating immunoglobulin E (IgE) phenotypes in 15 extended and 45 nuclear asthmatic families using a panel of 14 microsatellite markers. We examined the reported linkage between the TCR A/D locus on 14q11.2 and specific (cognate) allergic responses and observed supportive evidence for linkage between a general skin prick test reactivity trait (but not with total serum IgE) and TCRA microsatellite (in the total sample of informative sib-pairs p = 0.039, in selected sample of one or zero affected parent p = 0.017). We also show suggestive evidence for a novel linkage between markers D14S75 and D14S63 on 14q13-23 and log total serum IgE (p = 0.034 and p = 0.0029). The evidence for linkage with marker D14S63 on 14q23 is strengthened by the finding of association of allele 165 to log IgE (p = 0.0029). We conclude that chromosome 14q may contain a locus close to TCR A/D at 14q11.2 linked to skin prick reactivity and a locus at 14q13- 23 linked to total serum IgE.

Evidence for a role of HLA DRB1 alleles in the control of IgE levels, strengthened by interacting TCR A/D marker alleles.

BACKGROUND: MHC class II alleles at human chromosome 6p21.1 and alleles in the TCR A/D locus at human chromosome 14q11.2 have been implicated in susceptibility to specific allergies and the modulation of total serum IgE. It has also been hypothesized that HLA and TCR allelic interactions may have a strong influence on predisposition to allergic disease. OBJECTIVE: This study was performed to investigate the influence of HLA-DRB and DQB1 alleles and D14S50 alleles (adjacent to TCR A/D locus on 14q11.2), individually and in-combination, on total serum IgE levels, and on the development of specific allergies. METHODS: We performed an association study between HLA-DRB, HLA-DQB1 polymorphisms, D14S50 alleles, total serum IgE expression and specific allergies to house dust mite, grass pollens and cat fur. A sample of 181 individuals was drawn from a larger set of 2415 adults, sampled at random from a district in Nottingham. RESULTS: Strong association was observed between HLA-DRB1*0701 allele and high total serum IgE expression (P < 0.001). D14S50 alleles alone showed no evidence for independent association. However, there was a significant interaction between DRB1*0701 and D14S50 allele 170 such that, when both were present, there was a further increase in total serum IgE levels. CONCLUSION: This study suggests that DRB1*0701 allele is involved in the modulation of total serum IgE, and that there is an interaction between DRB1*0701 and a marker adjacent to TCR A/D in the control of IgE expression.

Association between - 308 tumour necrosis factor promoter polymorphism and bronchial hyperreactivity in asthma.

BACKGROUND: Tumour necrosis factor (TNF) is a pivotal cytokine in the inflammation underlying asthma. The TNF gene is located in the polymorphic HLA class 3 region on chromosome 6p. Several polymorphisms in this region have been described and associated with alteration of TNF secretion in vitro. OBJECTIVE: In this study we tested the hypothesis that two such polymorphisms, lymphotoxin alpha NcoI B*1 and -308 TNF2 may be components of the genetic predisposition to asthma. METHODS: Five hundred and fifty-six random individuals were studied, comprising approximately equal numbers of asthmatic subjects, with or without atopy, and a nonatopic nonasthmatic control group. In addition, 355 subjects (172 asthmatics) from 60 multiplex families were typed at the LTalpha NcoI locus. RESULTS: There was an association between allele two of the -308 TNF polymorphism and bronchial hyperreactivity (OR 2.12, 95% CI 1.04-4.32, P = 0.036). However, there was no association with LTalpha NcoI alleles. To determine whether this was influenced by linkage disequilibrium within the MHC, 91 subjects with bronchial hyperreactivity and 85 control subjects were typed for class 2 and 3 alleles. Following identification of the extended TNF2 haplotype, we found no independent association of these alleles with BHR. CONCLUSIONS: We conclude that the -308 TNF2 promoter polymorphism may form a component of the genetic predisposition to BHR in asthma.