Eric S. Silverman

Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial.

BACKGROUND The issue of whether regular use of an inhaled beta2-adrenergic agonist worsens airflow and clinical outcomes in asthma is controversial. Retrospective studies have suggested that adverse effects occur in patients with a genetic polymorphism that results in homozygosity for arginine (Arg/Arg), rather than glycine (Gly/Gly), at aminoacid residue 16 of the beta2-adrenergic receptor. However, the existence of any genotype-dependent difference has not been tested in a prospective clinical trial. METHODS Patients with mild asthma, not using a controller medication, were enrolled in pairs matched for forced expiratory volume in 1 s (FEV1) according to whether they had the Arg/Arg (n=37; four of 41 matches withdrew before randomisation) or Gly/Gly (n=41) genotype. Regularly scheduled treatment with albuterol or placebo was given in a masked, cross-over design, for 16-week periods. During the study, as-needed albuterol use was discontinued and ipratropium bromide was used as needed. Morning peak expiratory flow rate (PEFR) was the primary outcome variable. The primary comparisons were between treatment period for each genotype; the secondary outcome was a treatment by genotype effect. Analyses were by intention to treat. FINDINGS During the run-in period, when albuterol use was kept to a minimum, patients with the Arg/Arg genotype had an increase in morning PEFR of 23 L/min (p=0.0162); the change in patients with the Gly/Gly genotype was not significant (2 L/min; p=0.8399). During randomised treatment, patients with the Gly/Gly genotype had an increase in morning PEFR during treatment with regularly scheduled albuterol compared with placebo (14 L/min [95% CI 3 to 25]; p=0.0175). By contrast, patients with the Arg/Arg genotype had lower morning PEFR during treatment with albuterol than during the placebo period, when albuterol use was limited (-10 L/min [-19 to -2]; p=0.0209). The genotype-attributable treatment difference was therefore -24 L/min (-37 to -12; p=0.0003). There were similar genotype-specific effects in FEV1, symptoms, and use of supplementary reliever medication. INTERPRETATION Genotype at the 16th aminoacid residue of the beta2-adrenergic receptor affects the long-term response to albuterol use. Bronchodilator treatments avoiding albuterol may be appropriate for patients with the Arg/Arg genotype.

Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment.

Clinically similar asthma patients may develop airway obstruction by different mechanisms. Asthma treatments that specifically interfere with the 5-lipoxygenase (ALOX5) pathway provide a method to identify those patients in whom the products of the ALOX5 pathway (that is, the leukotrienes) contribute to the expression of the asthma phenotype. Failure of an asthma patient to respond to treatment with ALOX5-pathway modifiers indicates that leukotrienes are not critical to the expression of the asthmatic phenotype in that patient. We previously defined a family of DNA sequence variants in the core promoter of the gene ALOX5 (on chromosome 10q11.2) associated with diminished promoter-reporter activity in tissue culture. Because expression of ALOX5 is in part transcriptionally regulated, we reasoned that patients with these sequence variants may have diminished gene transcription, and therefore decreased ALOX5 product production and a diminished clinical response to treatment with a drug targeting this pathway. Such an effect indicates an interaction between gene promoter sequence variants and drug-treatment responses, that is, a pharmacogenetic effect of a promoter sequence on treatment responses.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

High-level expression of Egr-1 and Egr-1–inducible genes in mouse and human atherosclerosis

To understand the mRNA transcript profile in the human atherosclerotic lesion, RNA was prepared from the fibrous cap versus adjacent media of 13 patients undergoing carotid endarterectomy. cDNA expression arrays bearing 588 known genes indicated that lesions express unexpectedly high levels of the early growth response gene, Egr-1 (NGFI-A), a zinc-finger transcription factor that modulates a cluster of stress-responsive genes including PDGF and TGF-beta. Expression of Egr-1 was an average of 5-fold higher in the lesion than in the adjacent media, a result confirmed by RT-PCR, and many Egr-1-inducible genes were also strongly elevated in the lesion. Time-course analyses revealed that Egr-1 was not induced ex vivo. Immunocytochemistry indicated that Egr-1 was expressed prominently in the smooth muscle-actin positive cells, particularly in areas of macrophage infiltration, and in other cell types, including endothelial cells. Induction of atherosclerosis in LDL receptor-null mice by feeding them a high-fat diet resulted in a progressive increase in Egr-1 expression in the aorta. Thus, induction of Egr-1 by atherogenic factors may be a key step in coordinating the cellular events that result in vascular lesions.

Pathways of Egr-1-Mediated Gene Transcription in Vascular Biology

Vascular endothelium forms a continuous cellular interface between the circulating blood elements and the surrounding tissues. Endothelium forms a nonthrombogenic surface and selective permeability barrier capable of modulating vascular reactivity and blood flow. The integrity of the endothelium is fundamental for the maintenance of normal homeostasis. Injury to this lining results in dramatic changes in the functional characteristics of the endothelium, rendering it adhesive and prothrombotic. These changes may result from molecules inducibly expressed or simply secreted by injured endothelium. These initial events are correlated with subsequent inflammatatory and proliferative cellular changes associated with the development of vascular pathology.

TBX21: A functional variant predicts improvement in asthma with the use of inhaled corticosteroids

TBX21 encodes for the transcription factor T-bet (T-box expressed in T cells), which influences naïve T lymphocyte development and has been implicated in asthma pathogenesis. Specifically, the T-bet knockout mouse spontaneously develops airway hyperresponsiveness and other changes consistent with asthma. Because airway responsiveness is moderated by the use of inhaled corticosteroids in asthma, it is conceivable that genetic variation in TBX21 may alter asthma phenotypes in a treatment-specific fashion. Here we demonstrate that the nonsynonymous variation in TBX21 coding for replacement of histidine 33 with glutamine is associated with significant improvement in the PC20 (a measure of airway responsiveness) of asthmatic children in a large clinical trial spanning 4 years. We note that this increase occurs only in the children randomized to inhaled corticosteroids and that it dramatically enhances the overall improvement in PC20 associated with inhaled corticosteroid usage. The average PC20 at trial end for subjects on inhaled corticosteroids possessing a variant allele was in the normal range for nonasthmatics. In cellular models, we show that the TBX21 variant increases T helper 1 and decreases T helper 2 cytokine expression comparably with wild type. TBX21 may thus be an important determinant pharmacogenetic response to the therapy of asthma with inhaled corticosteroids.

The IL12B Gene Is Associated with Asthma

The IL12B gene on chromosome 5q31-33 encodes the p40 subunit of interleukin 12, an immunomodulatory cytokine. To test the hypothesis that the IL12B gene contains polymorphisms associated with asthma, we genotyped six haplotype-tagging polymorphisms in the IL12B gene, both in 708 children enrolled in the Childhood Asthma Management Program (CAMP) and in their parents. Using the family-based association test (FBAT) program and its haplotype (HBAT) and phenotype (PBAT) options, we tested each polymorphism and haplotype for association with asthma and asthma-related phenotypes. We tested positive associations for replication in a case-control study comparing 177 adult moderate-to-severe asthmatics with 177 nonasthmatic controls. In whites in the CAMP cohort, the A allele of the IL12B G4237A polymorphism was undertransmitted to asthmatic children (P=.0008, recessive model), the global test for haplotypes for affection status was positive (P=.009, multiallelic chi (2)), and two polymorphisms were associated with different atopy phenotypes. In addition, we found a strong association between the IL12B_4237 and IL12B_6402 polymorphisms and an asthma-severity phenotype in whites, which we also found in the independent population of white adult asthmatics. IL12B may be an important asthma gene.

IFN Regulatory Factor-1 Regulates IFN-γ-Dependent Cathepsin S Expression

Cathepsin S is a cysteine protease with potent endoproteolytic activity and a broad pH profile. Cathepsin S activity is essential for complete processing of the MHC class II-associated invariant chain within B cells and dendritic cells, and may also be important in extracellular matrix degradation in atherosclerosis and emphysema. Unique among cysteine proteases, cathepsin S activity is up-regulated by IFN-γ. Given its importance, we sought to elucidate the pathway by which IFN-γ increases cathepsin S expression. Our data demonstrate that the cathepsin S promoter contains an IFN-stimulated response element (ISRE) that is critical for IFN-γ-induced gene transcription in a cell line derived from type II alveolar epithelial (A549) cells. IFN response factor (IRF)-2 derived from A549 nuclear extracts associates with the ISRE oligonucleotide in gel shift assays, but is quickly replaced by IRF-1 following stimulation with IFN-γ. The time course of IRF-1/ISRE complex formation correlates with increased levels of IRF-1 protein and cathepsin S mRNA. Overexpression of IRF-1, but not IRF-2, markedly augments cathepsin S promoter activity in A549 cells. Furthermore, overexpression of IRF-1 increases endogenous cathepsin S mRNA levels in 293T epithelial cells. Finally, freshly isolated bone marrow cells from IRF-1−/− mice fail to up-regulate cathepsin S activity in response to IFN-γ. Thus, IRF-1 is the critical transcriptional mediator of IFN-γ-dependent cathepsin S activation. These data elucidate a new pathway by which IRF-1 may affect MHC class II processing and presentation.

Vascular smooth muscle cells express the transcriptional corepressor NAB2 in response to injury.

The early growth response 1 (Egr-1 or NGFI-A) gene product is a zinc finger protein transcription factor which has been implicated in the regulation of genes differentially expressed during the development of vascular disease. Egr-1 activity is regulated by alterations in the amount of protein, as well as protein-protein interactions with positive and negative transcriptional cofactors. NGFI-A-binding protein 2 (NAB2) is an example of a negative transcriptional cofactor capable of binding directly to Egr-1 and repressing Egr-1-mediated transcription. In this study, we show that NAB2 is rapidly and transiently expressed in vascular smooth muscle cells (VSMC) in response to the model agonist phorbol 12-myristate 13-acetate (PMA). This induction occurs at the protein as well as mRNA level, and the time course of induction trails closely behind that of Egr-1. NAB2 expression in VSMC is capable of inhibiting Egr-1 dependent gene expression in response to either PMA or fibroblastic growth factor-2 (FGF-2). In an in vivo model of mechanical arterial injury NAB2 levels also increase transiently in VSMC at a time when Egr-1 is elevated. It is possible that NAB2 is part of a negative-feedback mechanism which serves to down-regulate Egr-1-mediated gene transcription in injured VSMC.

Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma

BackgroundIdentifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. Signal transducer and activator of transcription 3 is a transcription factor latent in the cytoplasm; the gene (STAT3) is activated by a wide range of cytokines, and may play a role in lung development and asthma pathogenesis.MethodsWe genotyped six single nucleotide polymorphisms (SNPs) in the STAT3 gene in a cohort of 401 Caucasian adult asthmatics. The associations between each SNP and forced expiratory volume in 1 second (FEV1), as a percent of predicted, at the baseline exam were tested using multiple linear regression models. Longitudinal analyses involving repeated measures of FEV1 were conducted with mixed linear models. Haplotype analyses were conducted using imputed haplotypes. We completed a second association study by genotyping the same six polymorphisms in a cohort of 652 Caucasian children with asthma.ResultsWe found that three polymorphisms were significantly associated with baseline FEV1: homozygotes for the minor alleles of each polymorphism had lower FEV1 than homozygotes for the major alleles. Moreover, these associations persisted when we performed an analysis on repeated measures of FEV1 over 8 weeks. A haplotypic analysis based on the six polymorphisms indicated that two haplotypes were associated with baseline FEV1. Among the childhood asthmatics, one polymorphism was associated with both baseline FEV1 and the repeated measures of FEV1 over 4 years.ConclusionOur results indicate that genetic variants in STAT3, independent of asthma treatment, are determinants of FEV1 in both adults and children with asthma, and suggest that STAT3 may participate in inflammatory pathways that have an impact on level of lung function.