Penelope E. Graves

Association between genetic polymorphisms of the beta2-adrenoceptor and response to albuterol in children with and without a history of wheezing.

The beta2-adrenergic receptor (beta2AR) agonists are the most widely used agents in the treatment of asthma, but the genetic determinants of responsiveness to these agents are unknown. Two polymorphic loci within the coding region of the beta2AR have been recently described at amino acids 16 and 27. It has been reported that glycine at codon 16 (Gly-16) is associated with increased agonist-promoted downregulation of the beta2AR as compared with arginine-16 (Arg-16). The form of the receptor with glutamic acid at codon 27 (Glu-27), on the other hand, has been shown to be resistant to downregulation when compared with glutamine-27 (Gln-27), but only when coexpressed with Arg-16. To assess if different genotypes of these two polymorphisms would show differential responses to inhaled beta2AR agonists, we genotyped 269 children who were participants in a longitudinal study of asthma. Spirometry was performed before and after administration of 180 microg of albuterol, and a positive response was considered an increase of >15.3% predicted FEV1. There was marked linkage disequilibrium between the two polymorphisms, with 97.8% of all chromosomes that carried Arg-16 also carrying Gln-27. When compared to homozygotes for Gly-16, homozygotes for Arg-16 were 5.3 times (95% confidence interval 1.6-17.7) and heterozygotes for beta2AR-16 were 2.3 times (1.3-4.2) more likely to respond to albuterol, respectively. Similar trends were observed for asthmatic and nonasthmatic children, and results were independent of baseline lung function, ethnic origin, and previous use of antiasthma medication. No association was found between the beta2AR-27 polymorphism and response to albuterol. These results may explain some of the variability in response to therapeutic doses of albuterol in children.

Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations

Asthma is a common disease with a complex risk architecture including both genetic and environmental factors. We performed a meta-analysis of North American genome-wide association studies of asthma in 5,416 individuals with asthma (cases) including individuals of European American, African American or African Caribbean, and Latino ancestry, with replication in an additional 12,649 individuals from the same ethnic groups. We identified five susceptibility loci. Four were at previously reported loci on 17q21, near IL1RL1, TSLP and IL33, but we report for the first time, to our knowledge, that these loci are associated with asthma risk in three ethnic groups. In addition, we identified a new asthma susceptibility locus at PYHIN1, with the association being specific to individuals of African descent (P = 3.9 × 10−9). These results suggest that some asthma susceptibility loci are robust to differences in ancestry when sufficiently large samples sizes are investigated, and that ancestry-specific associations also contribute to the complex genetic architecture of asthma.

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

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

Resequencing Candidate Genes Implicates Rare Variants in Asthma Susceptibility

Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease.

cDNA and deduced amino acid sequences of a dog liver cytochrome P-450 of the IIIA gene subfamily

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

Integrated genome-wide association, coexpression network, and expression single nucleotide polymorphism analysis identifies novel pathway in allergic rhinitis

BackgroundAllergic rhinitis is a common disease whose genetic basis is incompletely explained. We report an integrated genomic analysis of allergic rhinitis.MethodsWe performed genome wide association studies (GWAS) of allergic rhinitis in 5633 ethnically diverse North American subjects. Next, we profiled gene expression in disease-relevant tissue (peripheral blood CD4+ lymphocytes) collected from subjects who had been genotyped. We then integrated the GWAS and gene expression data using expression single nucleotide (eSNP), coexpression network, and pathway approaches to identify the biologic relevance of our GWAS.ResultsGWAS revealed ethnicity-specific findings, with 4 genome-wide significant loci among Latinos and 1 genome-wide significant locus in the GWAS meta-analysis across ethnic groups. To identify biologic context for these results, we constructed a coexpression network to define modules of genes with similar patterns of CD4+ gene expression (coexpression modules) that could serve as constructs of broader gene expression. 6 of the 22 GWAS loci with P-value ≤ 1x10−6 tagged one particular coexpression module (4.0-fold enrichment, P-value 0.0029), and this module also had the greatest enrichment (3.4-fold enrichment, P-value 2.6 × 10−24) for allergic rhinitis-associated eSNPs (genetic variants associated with both gene expression and allergic rhinitis). The integrated GWAS, coexpression network, and eSNP results therefore supported this coexpression module as an allergic rhinitis module. Pathway analysis revealed that the module was enriched for mitochondrial pathways (8.6-fold enrichment, P-value 4.5 × 10−72).ConclusionsOur results highlight mitochondrial pathways as a target for further investigation of allergic rhinitis mechanism and treatment. Our integrated approach can be applied to provide biologic context for GWAS of other diseases.

Effect of quinine on digoxin kinetics

Six subjects were evaluated for the effect of quinine, the 1‐isomer of quinidine, on digoxin pharmacokinetics. A 1.0‐mg intravenous digoxin dose was given before and during quinine administration, followed by the measurement of digoxin serum and urine concentrations for 96 hr after each dose. Quinine reduced digoxin total body clearance by 26% from 2.98 to 2.22 ml/min/kg (p < 0.03). Digoxin elimination half‐life (t½) was lengthened from 34.2 to 51.8 hr, reflecting a 32% decrease in digoxin elimination rate constant (p < 0.003). Quinine did not reduce digoxin renal clearance or any volumes of distribution. The amount of digoxin excreted into the urine increased from x̄ = 628.29 μg to x̄ = 772.52 μg (p < 0.02). Digoxin nonrenal clearance decreased an average of 55% from 1.2 to 0.55 ml/min/kg (p < 0.05). These results suggest that quinine alters digoxin metabolism or biliary secretion, reducing digoxin total body clearance by a mechanism that is qualitatively similar, but quantitatively different, from quinidine.

Genetic Variation in Vascular Endothelial Growth Factor-A and Lung Function

RATIONALE Given the role of vascular endothelial growth factor (VEGF) in lung development, we hypothesized that polymorphisms in VEGF-A may be associated with lung function. OBJECTIVES The current study was designed to assess the role of genetic variants in VEGF-A as determinants of airway function from infancy through early adulthood. METHODS Association between five single-nucleotide polymorphisms (SNPs) in VEGF-A and lung function were assessed longitudinally in two unselected birth cohorts and cross-sectionally among infants. Replication with two SNPs was conducted in adults and children with asthma. We investigated the functionality of the SNP most consistently associated with lung function (rs3025028) using Western blotting to measure the ratio of plasma VEGF-A(165b)/panVEGF-A(165) among homozygotes. MEASUREMENTS AND MAIN RESULTS In two populations in infancy, C-allele homozygotes of rs3025028 had significantly higher VmaxFRC, forced expiratory flow(50), and forced expiratory flow(25-75) compared with other genotype groups. Among preschool children (age 3 yr), C allele of rs3025028 was associated with significantly higher specific airway conductance, with similar findings observed for lung function in school-age children. For FEV(1)/FVC ratio similar findings were observed among adolescents and young adults (birth cohort), and then replicated in adults and schoolchildren with asthma (cross-sectional studies). For rs3025038, plasma VEGF-A(165b)/panVEGF-A(165) was significantly higher among CC versus GG homozygotes (P ≤ 0.02) at birth, in school-age children, and in adults. CONCLUSIONS We report significant associations between VEGF-A SNP rs3025028 and parameters of airway function measured throughout childhood, with the effect persisting into adulthood. We propose that the mechanism may be mediated through the ratios of active and inhibitory isoforms of VEGF-A(165), which may be determined by alternative splicing.

Ethnic-specific associations of rare and low-frequency DNA sequence variants with asthma

Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (<1%) and low-frequency (1–5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls and 590 case–parent trios representing European Americans, African Americans/African Caribbeans and Latinos. Our study reveals one low-frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample (P=4.31 × 10−6; OR=1.25; MAF=1.21%) and two genes harbouring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12–21 asthma locus in the Latino and combined samples (P=7.81 × 10−8 and 4.09 × 10−8, respectively) and MTHFR in the African ancestry sample (P=1.72 × 10−6). Our results suggest that associations with rare and low-frequency variants are ethnic specific and not likely to explain a significant proportion of the ‘missing heritability’ of asthma.

Digitoxin-quinidine interaction: pharmacokinetic evaluation.

The effect of quinidine on digitoxin single-dose pharmacokinetics was evaluated in five healthy adults. Blood was collected for 3 weeks, and a complete urine collection was obtained for 4 days, after a single intravenous dose of digitoxin. The protocol was conducted once while each subject was taking oral quinidine, for 3 weeks, and then repeated 10 days after discontinuing quinidine treatment. Quinidine induced the following changes in digitoxin pharmacokinetics: Elimination half-life was prolonged from 174 +/- 25 to 261 +/- 58 hours (p less than 0.02); total body clearance decreased from 1.54 +/- 0.40 to 1.09 +/- 0.31 mL/h . kg (p less than 0.05); renal clearance decreased from 0.65 +/- 0.07 to 0.46 +/- 0.17 mL/h . kg (p less than 0.05). Digitoxin volume of distribution and protein binding were unaltered by quinidine. Quinidine caused a rise in serum digitoxin levels. Digitoxin total body clearance was decreased by quinidine to an extent comparable to that reported for digoxin; however, the mechanism of the interaction with the two digitalis glycosides may, in part, be different.