Amanda Wheatley

Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of inflammatory responses and thus have important roles in the pathogenesis of inflammatory diseases. Here we describe a genome-wide association scan for sequence variants affecting eosinophil counts in blood of 9,392 Icelanders. The most significant SNPs were studied further in 12,118 Europeans and 5,212 East Asians. SNPs at 2q12 (rs1420101), 2q13 (rs12619285), 3q21 (rs4857855), 5q31 (rs4143832) and 12q24 (rs3184504) reached genome-wide significance (P = 5.3 × 10−14, 5.4 × 10−10, 8.6 × 10−17, 1.2 × 10−10 and 6.5 × 10−19, respectively). A SNP at IL1RL1 associated with asthma (P = 5.5 × 10−12) in a collection of ten different populations (7,996 cases and 44,890 controls). SNPs at WDR36, IL33 and MYB that showed suggestive association with eosinophil counts were also associated with atopic asthma (P = 4.2 × 10−6, 2.2 × 10−5 and 2.4 × 10−4, respectively). We also found that a nonsynonymous SNP at 12q24, in SH2B3, associated significantly (P = 8.6 × 10−8) with myocardial infarction in six different populations (6,650 cases and 40,621 controls).

Association of Glu 27 β2-adrenoceptor polymorphism with lower airway reactivity in asthmatic subjects

Cells expressing the Glu 27 beta 2-adrenoceptor polymorphism show attenuated down-regulation of the receptor after long-term exposure to agonist. We studied beta 2-adrenoceptor genotype for the Gln/Glu 27 polymorphism and airway reactivity in 65 patients with mild to moderate asthma. Glu 27 homozygotes had a four-fold higher geometric mean methacholine PD20 (provocative dose) than individuals who were homozygous for the wild-type (Gln 27) form of the receptor; heterozygotes had an intermediate value (3.23, 0.86, 1.96 mumol, respectively). These data suggest that beta 2-adrenoceptor genotype is important in the establishment of the asthmatic phenotype.

The glutamine 27 β2-adrenoceptor polymorphism is associated with elevated IgE levels in asthmatic families

BACKGROUND The beta2-adrenoceptor polymorphisms occurring at amino acid positions 16 (arginine to glycine) and 27 (glutamine to glutamate) are known to be functionally relevant and also disease-modifying in subjects with asthma. However, the contribution of these polymorphisms to the development of the asthmatic phenotype or other markers for allergic disease remains to be established. OBJECTIVE This large family study examines the contributions of these polymorphisms in determining the heritable component of markers for allergic disease in asthmatic families. METHODS Three hundred twenty-four individuals from 60 families multiplex for asthma selected by means of an asthmatic proband were characterized for the following markers of allergic disease: asthma, atopy, and serum IgE. The polymerase chain reaction was used to generate a 234 base pair fragment spanning the region of interest, and the beta2-adrenoceptor polymorphism was then defined by allele-specific oligonucleotide hybridization. Segregation analysis was then performed. RESULTS We found a significant association (p = 0.009) between the glutamine 27 beta2-adrenoceptor polymorphism and elevated levels of IgE, which was supported by the observation of linkage between IgE and beta2-adrenoceptor polymorphisms at locus 27 (p = 0.037). However, there was no association between either the arginine-glycine 16 or the glutamine-glutamate 27 beta2-adrenoceptor polymorphism and an increased risk of asthma or atopy per se. CONCLUSION The glutamine 27 beta2-adrenoceptor polymorphism appears to contribute to IgE variability in families with asthma. However, it seems that although both amino acid 16 and 27 beta2-adrenoceptor polymorphisms are disease-modifying in subjects with asthma, they do not contribute markedly to the development of the asthmatic phenotype.

β2-Adrenoceptor Polymorphism Determines Vascular Reactivity in Humans

Altered &bgr;-adrenergic regulation has been reported in individuals with hypertension. The variability in vascular responsiveness to &bgr;-agonists, such as isoproterenol, observed in humans may be explained partially by &bgr;2-adrenoceptor polymorphism. Individuals with the Gln27 form of the receptor may show reduced vascular reactivity because of downregulation expression of the receptor in the vasculature. We screened 127 normotensive white subjects, 37 of whom were homozygous for these alleles. Thirty-two subjects (17 Gln27 and 15 Glu27) agreed to receive brachial artery infusions of isoproterenol at doses of 1 to 300 ng · min−1; forearm blood flow was measured by using venous occlusion plethysmography. Of these subjects, 25 (12 Glu27 and 13 Gln27) received local doses of isoproterenol (0.3 to 30.0 ng · min−1) via a dorsal hand vein preconstricted with norepinephrine. Compared with subjects homozygous for the Glu27 allele, subjects with the Gln27 substitution had lower baseline blood flow and, in response to isoproterenol, had a significantly attenuated increase in forearm blood flow. This pattern was more marked in veins. We also studied the relationship between the position 16 polymorphism and vascular reactivity. Homozygotes for Arg16 had significantly lower basal blood flow and attenuated increases in forearm blood flow compared with the Gly16 homozygotes. This was significant in veins but not in arteries. Thus, &bgr;2-adrenoceptor genotype determines vascular responses to isoproterenol in forearm resistance vessels and in capacitance vessels. Further studies are necessary to establish whether &bgr;2-adrenoceptor polymorphisms are important in the genesis of hypertension.

Identification of novel polymorphisms within the promoter region of the human β2 adrenergic receptor gene

By screening the 1470 bp 5′ to the start codon of the human β2 adrenergic receptor gene, we have identified a total of eight polymorphisms (−20 T→C, −47 T→C, −367 T→C, −468 C→G, −654 G→A, −1023 G→A, −1343 A→G and −1429 T→A c.f. β2 adrenergic receptor start codon). Transient transfection of 5′ flanking deletion luciferase reporter constructs demonstrated the majority of activity of the human β2 adrenergic gene 5′ flanking region to be present within a 549 bp fragment immediately upstream from the start codon. Because of linkage disequilibrium, some combinations of polymorphisms were particularly frequent. We transiently transfected COS‐7 cells with luciferase constructs under the control of the 549 bp of 5′ flanking DNA containing the two most frequent extended haplotypes in this region. Luciferase activity was significantly reduced in cells transfected with the ‘mutant’ construct (−20C, −47C, −367C, −468G) c.f. the ‘wild‐type’ construct (−20T, −47T, −367T, −468C). These data suggest that polymorphisms have the potential to alter human β2 adrenergic receptor gene expression.

Association of CCR5 ▵32 with reduced risk of asthma

Summary We report that individuals carrying the CCR5▵32 mutation, a naturally occurring variant of the C-C chemokine receptor 5 (CCR5), are at reduced risk of developing asthma. These data suggest a possible explanation for the high prevalence of this mutation in the general population.

β2-adrenoceptor polymorphisms and asthma from childhood to middle age in the British 1958 birth cohort: a genetic association study

BACKGROUND Functionally relevant polymorphisms of the beta2-adrenoceptor gene (ADRB2) are common in white populations, but their contribution to the burden of airways disease in the population is uncertain. We aimed to relate the long-term prevalence of asthma or wheeze to functional coding region polymorphisms in the ADRB2 gene. METHODS The British 1958 birth cohort consisted of all people born in Britain during a week in 1958. Asthma, wheezy bronchitis, and wheezing were ascertained by interview at ages 7, 11, 16, 23, 33, and 42 years, and lung function tests at 35 and 45 years. DNA samples from 8018 participants in the 45-year follow-up were genotyped for three coding variants in the ADRB2 gene. We extend the follow-up of this nationwide cohort by a further 10 years and relate asthma prevalence, prognosis, and lung function to functional coding region polymorphisms in the ADRB2 gene in the cohort members who contributed DNA samples. We also compared and combined our findings with those reaching significance in two previous meta-analyses. FINDINGS Half the cohort (4105 of 8018) had some history of wheezing illness by age 42 years. Neither lifetime prevalence nor age at onset were related to ADRB2 coding variants. However, the common polymorphisms Arg16Gly (rs1042713, Arg 16 allele frequency 36.3%) and Gln27Glu (rs1042714, Glu 27 allele frequency 44.6%) were significantly associated with persistence of asthmatic symptoms from childhood to middle age. Among homozygotes for the Arg16-Gln27 haplotype at these loci, 19.3% (41 of 212) childhood wheezers had five or more wheezing episodes in the past year at age 42, compared with 11.9% (71 of 599) with no copy of this haplotype. However, only 3% of all frequent adult wheezing was statistically attributable to this haplotype. The less common Thr164Ile polymorphism (rs1800888, Ile allele frequency 1.5%) was not a major predictor of either frequency or prognosis of asthma. Our data do not support the findings of previous meta-analyses when considered in isolation or when combined with their contributory studies. INTERPRETATION ADRB2 polymorphisms might predict a small component of the long-term prognosis in childhood asthma, but are not important determinants of asthma incidence or prevalence in the British population.

Contribution of ADAM33 polymorphisms to the population risk of asthma

Background:ADAM 33 is the first gene identified as a candidate for asthma by positional cloning techniques, with association studies reaching impressive statistical significance. It has a postulated role in myogenesis, airway modelling, and signalling via protein shedding. Concerns over the methodology of the initial study have led to several attempts at replication, with inconsistent results. Method: To clarify the role of ADAM33 in determining the risk of asthma in the general population, new transmission disequilibrium and case-control studies were undertaken followed by a meta-analysis of all existing data. Results: Studies in Icelandic and UK populations revealed no association when taken in isolation. The meta-analysis, however, showed that the F+1 and ST+7 variants were significantly associated with asthma in both types of study. Conclusions: The additional risk imparted by this variation would account for 50 000 excess asthma cases in the UK alone. This study also demonstrates the size of study required to investigate such hypotheses adequately.

Association analysis of β2 adrenoceptor polymorphisms with hypertension in a Black African population

Objective To determine whether or not β2 adrenoceptor polymorphism is a risk factor for the development of hypertension in a Black South African population Background Attenuated vasodilator responses to endogenous catecholamines may contribute to the aetiology of hypertension. Downregulation of β2 adrenoreceptors (β2AR) following stimulation with agonists is determined in part by variation at the β2AR gene locus. The Glu27 β2AR genotype results in attenuated downregulation compared with the wild-type Gln27 receptor, whereas Gly16 exhibits enhanced down-regulation compared to Arg16. Possible racial differences in the prevalence of the β2AR polymorphisms may be an explanation for the blunted responses to isoprenaline and the increased prevalence of hypertension in Black African populations. Methods One hundred and ninety-two unrelated hypertensives and 123 normotensives of Black South African origin were studied. Hypertensives were recruited from hospital hypertension clinics in the province of Gauteng and if on treatment, had a 2–4 week washout period before 24-h ambulatory blood pressure assessment. Normotensive controls were recruited from the same community. Results There was no significant association between either the Arg–Gly16 polymorphism or the Gln–Glu27 polymorphism and hypertension status. Furthermore, in the hypertensives, no significant association was seen between β2AR genotype at either site and clinical bloodpressure, 24-h blood pressure or left ventricular mass. A significant association was seen between Arg16 homozygotes and lower body mass index in hypertensives (P = 0.007) although this was not a primary end point. Interestingly, the Glu27 polymorphism was much rarer in this population (allelic frequency 17%) compared to a Caucasian population. Conclusion These data suggest that β2AR polymorphism is not a risk factor for hypertension per se in this defined population. The possibility that the decreased prevalence of Glu27 in black South African populations explains blunted vasodilator responses to isoprenaline requires further study.

Beta2 adrenoceptor promoter polymorphisms: extended haplotypes and functional effects in peripheral blood mononuclear cells.

Background: The β2 adrenoceptor and its 5′ untranslated region contain a number of genetic variants. The aim of this study was to investigate the potential for genetic variation at this locus to influence the expression of β2 adrenoceptors on circulating peripheral blood mononuclear cells (PBMCs). Methods: Genotype was determined in 96 individuals with asthma for four polymorphisms at the β2 adrenoceptor locus. β2 adrenoceptor binding and cyclic AMP responses to isoprenaline in PBMCs were determined and the relationship between genotype/haplotype and β2 adrenoceptor expression and response to isoprenaline examined. Results: β2 adrenoceptor promoter polymorphisms were found to be common in white subjects. Strong linkage disequilibrium exists across this locus, resulting in the occurrence of several common haplotypes. No single polymorphism or haplotype was correlated with the level of β2 adrenoceptor expression or cyclic AMP responses to isoprenaline in vitro. Conclusion: β2 adrenoceptor polymorphisms, when considered in isolation or by extended haplotypes, do not determine the basal level of expression or coupling of β2 adrenoceptors in PBMCs from asthmatic subjects.