Audrey Poon

Asthma and genes encoding components of the vitamin D pathway

BackgroundGenetic variants at the vitamin D receptor (VDR) locus are associated with asthma and atopy. We hypothesized that polymorphisms in other genes of the vitamin D pathway are associated with asthma or atopy.MethodsEleven candidate genes were chosen for this study, five of which code for proteins in the vitamin D metabolism pathway (CYP27A1, CYP27B1, CYP2R1, CYP24A1, GC) and six that are known to be transcriptionally regulated by vitamin D (IL10, IL1RL1, CD28, CD86, IL8, SKIIP). For each gene, we selected a maximally informative set of common SNPs (tagSNPs) using the European-derived (CEU) HapMap dataset. A total of 87 SNPs were genotyped in a French-Canadian family sample ascertained through asthmatic probands (388 nuclear families, 1064 individuals) and evaluated using the Family Based Association Test (FBAT) program. We then sought to replicate the positive findings in four independent samples: two from Western Canada, one from Australia and one from the USA (CAMP).ResultsA number of SNPs in the IL10, CYP24A1, CYP2R1, IL1RL1 and CD86 genes were modestly associated with asthma and atopy (p < 0.05). Two-gene models testing for both main effects and the interaction were then performed using conditional logistic regression. Two-gene models implicating functional variants in the IL10 and VDR genes as well as in the IL10 and IL1RL1 genes were associated with asthma (p < 0.0002). In the replicate samples, SNPs in the IL10 and CYP24A1 genes were again modestly associated with asthma and atopy (p < 0.05). However, the SNPs or the orientation of the risk alleles were different between populations. A two-gene model involving IL10 and VDR was replicated in CAMP, but not in the other populations.ConclusionA number of genes involved in the vitamin D pathway demonstrate modest levels of association with asthma and atopy. Multilocus models testing genes in the same pathway are potentially more effective to evaluate the risk of asthma, but the effects are not uniform across populations.

Pathogenesis of severe asthma

Patients with severe asthma have asthma symptoms which are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction. Epidemiological and clinical evidences point to the fact that severe asthma is not a single phenotype. Cluster analyses have identified subclasses of severe asthma using parameters such as patient characteristics, and cytokine profiles have also been useful in classifying moderate and severe asthma. The IL‐4/IL‐13 signalling pathway accounts for the symptoms experienced by a subset of severe asthmatics with allergen‐associated symptoms and high serum immunoglobulin E (IgE) levels, and these patients are generally responsive to anti‐IgE treatment. The IL‐5/IL‐33 signalling pathway is likely to play a key role in the disease pathogenesis of those who are resistant to high doses of inhaled corticosteroid but responsive to systemic corticosteroids and anti‐IL5 therapy. The IL‐17 signalling pathway is thought to contribute to ‘neutrophilic asthma’. Although traditionally viewed as players in the defence mechanism against viral and intracellular bacterial infection, mounting evidence supports a role for Th1 cytokines such as IL‐18 and IFN‐γ in severe asthma pathogenesis. Furthermore, these cytokine signalling pathways interact to contribute to the spectrum of clinical pathological outcomes in severe asthma. To date, glucocorticoids are the most effective anti‐asthma drugs available, yet severe asthma patients are typically resistant to the effects of glucocorticoids. Glucocorticoid receptor dysfunction and histone deacetylase activity reduction are likely to contribute to glucocorticoid resistance in severe asthma patients. This review discusses recent development in different cytokine signalling pathways, their interactions and steroid resistance, in the context of severe asthma pathogenesis.

Genetic and histologic evidence for autophagy in asthma pathogenesis

To the Editor: Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxidative species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.(1) Autophagy, the process of cellular waste disposal through lysosomal -dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.(2) Although autophagy is augmented in the lungs of COPD patients compared to healthy control subjects (3), evidence for autophagy in asthma, particularly moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1(ULK1), sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), beclin 1 (BECN1) and autophagy related 5 homolog (ATG5)). (2) We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (The Saguenay-Lac-Saint-Jean (SLSJ) asthma Study) using the family based association test (FBAT) statistic and UNPHASED software for an odds ratio estimate.(4, 5) Patient recruitment has previously been described.(6) These SNPs have been genotyped previously in a genome-wide association study.(6) To reduce the likelihood of false positive findings, we reset the statistical significance threshold from p=0.05 to p=0.001 according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family based population; the non-Hispanic Caucasian participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described. (7) The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects gave informed consents. In the SLSJ asthma study a total of 1338 individuals (483 nuclear families) with known asthma status were included in the analysis, and 336 individuals were either probands or affected siblings. Of this group, the male:female ratio was 0.83. The mean age was 16.45 (standard deviation (SD) +/− 9.43) years. 77.1% were atopic. The mean (SD) forced expiratory volume in 1 second (FEV1)% predicted was 94.1(20.1)%. A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table 1). Allele G with allele frequency of 0.88 is over - transmitted to asthmatic offspring (p=0.0002), (Odds ratio = 1.35, (95% confidence interval = 1.01 and 1.89). SNP rs12212740 was not associated with asthma in CAMP, however, it was associated with pre - bronchodilator FEV1 (pre-FEV1) (adjusted for age, sex and height) (p=0.04). In the SLSJ cohort, rs12212740 was associated with pre-FEV1 (p=0.007). In both populations, allele G was negatively associated with adjusted pre-FEV1. SNP rs12212740 is located in intron 3 of ATG5, 7kb downstream and 8kb upstream of exon 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and pre-FEV1 in both study populations suggest that autophagy is associated with reduced lung function in asthmatic subjects. Table 1 Association of SNPs and asthma in the SLSJ asthma study To determine if autophagy is present in the airways of asthmatic individuals, bronchial biopsy stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherche en Sante du Quebec (McGill University Health Centre site) were obtained. Patient recruitment and bronchoscopy have previously been described.(8) Bronchial biopsy tissue from a moderately severe asthma patient and a healthy control were viewed by electron microscopy (EM) for double membrane autophagosomes. Here, we demonstrated by EM in a tissue sample from a moderately severe asthma subject evidence of autophagy in asthma pathogenesis. Using EM, double membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a moderate asthma subject is depicted in Figures 1A-C, and epithelial cells in figures 2A-C. Corresponding fibroblast and epithelial cells from a healthy control are depicted in figures 1D-F and figures 2D-F, respectively, where fewer or no autophagosome was detected. Fig 1 A double membrane autophagosome was detected in a fibroblast from a bronchial biopsy tissue sample of a moderate asthma subject. Tissue was viewed at magnifications of (A) 1480x, (B) 16100x and (C) 62200x. Boxed areas were viewed under higher magnification ... Fig 2 Autophagosomes were detected in bronchial epithelial cells from a moderate asthma subject. Tissue was viewed at magnifications of (A) 4030x, (B) 9760x and (C) 37000x, respectively. Corresponding epithelial cells of a healthy subject was viewed at magnifications ... This is the first report to present genetic and histological evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1 and the complexes are found on the outer membrane of the forming autophagosome.(2) We speculate that the positive association of allele G with asthma and the negative association with pre-FEV1 in asthmatics may be due to the inverse relationship between pre-FEV1 and asthma severity(9). If allele G is a risk factor for low pre-FEV1, given that pre-FEV1 tends to be lower in those with more severe forms of asthma, the allele would also increase the risk of developing moderate-to-severe asthma. The genetic association of ATG with pre-FEV1 in asthmatics and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a moderately severe asthma patient suggest an association between autophagy and reduced lung function in asthmatic subjects. At present the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis.

Asthma phenotypes and endotypes.

Purpose of review It is increasingly clear that asthma is not a single disease, but a disorder with vast heterogeneity in pathogenesis, severity, and treatment response. In this review, we discuss the present understanding of different asthma phenotypes and endotypes, and the prospects of personalized medicine for asthma. Recent findings The recognition of diverse biological backgrounds in which asthma, and particularly severe asthma, can manifest has prompted the search for refined phenotypes and endotypes in asthma. Such appreciation of the heterogeneity in asthma is also prompting clinical trials to focus on specific subgroups of asthma, as demonstrated by the clinical trial of lebrikizumab. Summary Patients with severe asthma have asthma symptoms that are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction even with aggressive therapy. Although asthma is traditionally viewed as an eosinophilic inflammatory disorder associated with a T-helper cell type 2 (Th2) immune response, recent studies have identified involvement of other effector cells, nonclassical Th2 cytokines and non-Th2 cytokines in severe asthma pathogenesis. Results of several clinical trials of anticytokine antibodies demonstrated the effectiveness of tailoring asthma treatment on the basis of an individuals biology.

Association of corticotropin-releasing hormone receptor-2 genetic variants with acute bronchodilator response in asthma.

Objective Corticotropin-releasing hormone receptor (CRHR)-2 participates in smooth muscle relaxation response and may influence acute airway bronchodilator response to short-acting &bgr;2-agonist treatment of asthma. We aim to assess associations between genetic variants of CRHR2 and acute bronchodilator response in asthma. Methods We investigated 28 single nucleotide polymorphisms in CRHR2 for associations with acute bronchodilator response to albuterol in 607 Caucasian asthmatic patients recruited as part of the Childhood Asthma Management Program. Replication was conducted in two Caucasian adult asthma cohorts – a cohort of 427 participants enrolled in a completed clinical trial conducted by Sepracor Inc. (Massachusetts, USA) and a cohort of 152 participants enrolled in the Clinical Trial of Low-Dose Theophylline and Montelukast conducted by the American Lung Association Asthma Clinical Research Centers. Results Five variants were significantly associated with acute bronchodilator response in at least one cohort (P≤0.05). Variant rs7793837 was associated in Childhood Asthma Management Program and Low-Dose Theophylline and Montelukast (P=0.05 and 0.03, respectively) and haplotype blocks residing at the 5′ end of CRHR2 were associated with response in all three cohorts. Conclusion We report for the first time, at the gene level, replicated associations between CRHR2 and acute bronchodilator response. Although no single variant was significantly associated in all three cohorts, the findings that variants at the 5′ end of CRHR2 are associated in each of three cohorts strongly suggest that the causative variants reside in this region and its genetic effect, although present, is likely to be weak.

ATG5, autophagy and lung function in asthma

Reactive oxidative species (ROS) are essential in cellular survival; however, excessive production and chronic exposure to ROS pose serious health threats. Excessive production of ROS is thought to play a pivotal role in the pathogenesis of asthma, where exhaled levels of ROS have been found to positively correlate with disease severity. Autophagy is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage, and presents itself as a good candidate pathway for investigation in asthma pathogenesis. Given the role of oxidative stress in the pathogenesis of asthma and disease severity, we hypothesized that autophagy is associated with asthma pathogenesis, and sought to detect its presence using both genetic and histological approaches. We found variant rs12212740, an intronic SNP of ATG5, to be associated with asthma and forced expiratory volume in 1 second (FEV1) percent predicted in the French Canadian population and with FEV1 in an American Caucasian cohort. Furthermore, double-membrane autophagosomes were more easily detected in fibroblast and epithelial cells from a bronchial biopsy tissue of a moderately severe asthma patient compared with corresponding cells of a healthy subject. Asthma is associated with a cytokine milieu [e.g., interleukin (IL)-13] that promotes transforming growth factor-β1 (TGFβ1) affiliated airway remodeling, and agonistic relationships existed among these cytokines and ROS. Hence, autophagy may be a cellular mechanism that promotes TGFβ1 airway remodeling and loss of lung function in asthma.

Vitamin D deficiency and severe asthma.

Vitamin D has received tremendous amount of attention recently due to the ever-increasing reports of association between vitamin D deficiency and a wide range of conditions, from cancer to fertility to longevity. The fascination of disease association with vitamin D deficiency comes from the relatively easy solution to overcome such a risk factor, that is, either by increase in sun exposure and/or diet supplementation. Many reviews have been written on a protective role of vitamin D in asthma and related morbidities; here, we will summarize the epidemiological evidence supporting a role of vitamin D against hallmark features of severe asthma, such as airway remodeling and asthma exacerbations. Furthermore, we discuss data from in vitro and in vivo studies which provide insights on the potential mechanisms of how vitamin D may protect against severe asthma pathogenesis and how vitamin D deficiency may lead to the development of severe asthma. Approximately 5-15% of asthmatic individuals suffer from the more severe forms of disease in spite of aggressive therapies and they are more likely to have irreversible airflow obstruction associated with airway remodeling. At present drugs commonly used to control asthma symptoms, such as corticosteroids, do not significantly reverse or reduce remodeling in the airways. Hence, if vitamin D plays a protective role against the development of severe asthma, then the most effective therapy may simply be a healthy dose of sunshine.

Postural changes in blood pressure associated with interactions between candidate genes for chronic respiratory diseases and exposure to particulate matter.

Background Fine particulate matter [aerodynamic diameter ≤ 2.5 μm (PM2.5)] has been associated with autonomic dysregulation. Objective We hypothesized that PM2.5 influences postural changes in systolic blood pressure (ΔSBP) and in diastolic blood pressure (ΔDBP) and that this effect is modified by genes thought to be related to chronic lung disease. Methods We measured blood pressure in participants every 3–5 years. ΔSBP and ΔDBP were calculated as sitting minus standing SBP and DBP. We averaged PM2.5 over 48 hr before study visits and analyzed 202 single nucleotide polymorphisms (SNPs) in 25 genes. To address multiple comparisons, data were stratified into a split sample. In the discovery cohort, the effects of SNP × PM2.5 interactions on ΔSBP and ΔDBP were analyzed using mixed models with subject-specific random intercepts. We defined positive outcomes as p < 0.1 for the interaction; we analyzed only these SNPs in the replicate cohort and confirmed them if p < 0.025 with the same sign. Confirmed associations were analyzed within the full cohort in models adjusted for anthropometric and lifestyle factors. Results Nine hundred forty-five participants were included in our analysis. One interaction with rs9568232 in PHD finger protein 11 (PHF11) was associated with greater ΔDBP. Interactions with rs1144393 in matrix metalloprotease 1 (MMP1) and rs16930692, rs7955200, and rs10771283 in inositol 1,4,5-triphosphate receptor, type 2 (ITPR2) were associated with significantly greater ΔSBP. Because SNPs associated with ΔSBP in our analysis are in genes along the renin–angiotensin pathway, we then examined medications affecting that pathway and observed significant interactions for angiotensin receptor blockers but not angiotensin-converting enzyme inhibitors with PM2.5. Conclusions PM2.5 influences blood pressure and autonomic function. This effect is modified by genes and drugs that also act along this pathway.

Very important pharmacogene summary for VDR

The vitamin D receptor (VDR) binds the active form of vitamin D (1,25-dihydroxyvitamin D3). It belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. The gene was cloned by Baker and colleagues in 1988 and maps to chromosome 12q13.11 [1]. It consists of nine exons with at least six isoforms of exon 1, spans 63.5 kb, and encodes a 427 amino acid protein [2]. Alternative splicing results in multiple transcript variants encoding the VDR protein of different lengths [3,4]. The interaction of 1,25-dihydroxyvitamin D3 with VDR modulates many biological activities of the neural, immune, and endocrine systems, including calcium and phosphorous homeostasis, apoptosis, and cell differentiation (reviewed by Hewison and colleagues [5-7]). Its pleiotropic property is reflected by the findings that VDR is expressed in at least 37 tissues, which can roughly be grouped into seven biological systems (calcium homeostasis, immune, pancreatic β cells, muscle, cardiovascular, brain, and lung) [8-10]. The most characterized mechanism is the binding of vitamin D3 to nuclear VDR, which activates the receptor to form a heterodimer with the retinoid-X receptor and interacts with a specific DNA sequence on the gene promoter regions called the ‘vitamin D response element’ (VDRE). Transcription repressors occupying the VDRE are then replaced by transcription activators to initiate transcription of targeted genes. Microarray analyses using different human cell lines have identified over 100 genes with VDREs in the promoter regions, all potential targets of the 1,25-dihydroxyvitamin D3–VDR complex [11-13]. In human oral squamous carcinoma SCC25 cells, genes with characterized VDREs and of which demonstrated greater than 10-fold increase after treatment with 1,25-dihydroxy-vitamin D3 include 24-hydroxylase (CYP24), 17β-hydroxy-steroid dehydrogenase (HSD17B2), CD14 (CD14), type XIII collagen (COL13A1), and 5-lipoxygenase (ALOX5) [13]. The effect of 1,25-dihydroxyvitamin D3 by binding to VDR in the nucleus is often described as the slow-acting genomic effect; the effect will take days or hours to be noticed. Alternatively, 1,25-dihydroxyvitamin D3 can bind to VDR located in the caveolae in the plasma membrane to exert rapid responses through production of second messengers [14]. Pathways that have been shown to be activated by the 1,25-dihydroxyvitamin D3–VDR[memb] complex include mitogen-activated protein kinase-dependent [15], cAMP-dependent [16], phospholipase A2-dependent [17], phospholipase C-dependent [18], phosphatidylinositol 3-kinase-dependent [19], and protein kinase C-dependent [20] pathways. Examples of such rapid response include rapid Ca2+ absorption in the duodenum [21,22], opening of Cl− channels and secretion in osteoblasts, insulin secretion from pancreatic β-cells [23,24], and vascular smooth muscle cell migration [19].

Association of variants in innate immune genes with asthma and eczema

To cite this article: Sharma S, Poon A, Himes BE, Lasky‐Su J, Sordillo JE, Belanger K, Milton DK, Bracken MB, Triche EW, Leaderer BP, Gold DR, Litonjua AA. Association of variants in innate immune genes with asthma and eczema. Pediatric Allergy Immunology 2012: 23: 315–323.