Catherine M. Hayden

Identification of IL6R and chromosome 11q13.5 as risk loci for asthma

BACKGROUND We aimed to identify novel genetic variants affecting asthma risk, since these might provide novel insights into molecular mechanisms underlying the disease. METHODS We did a genome-wide association study (GWAS) in 2669 physician-diagnosed asthmatics and 4528 controls from Australia. Seven loci were prioritised for replication after combining our results with those from the GABRIEL consortium (n=26,475), and these were tested in an additional 25,358 independent samples from four in-silico cohorts. Quantitative multi-marker scores of genetic load were constructed on the basis of results from the GABRIEL study and tested for association with asthma in our Australian GWAS dataset. FINDINGS Two loci were confirmed to associate with asthma risk in the replication cohorts and reached genome-wide significance in the combined analysis of all available studies (n=57,800): rs4129267 (OR 1·09, combined p=2·4×10(-8)) in the interleukin-6 receptor (IL6R) gene and rs7130588 (OR 1·09, p=1·8×10(-8)) on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene (LRRC32, also known as GARP). The 11q13.5 locus was significantly associated with atopic status among asthmatics (OR 1·33, p=7×10(-4)), suggesting that it is a risk factor for allergic but not non-allergic asthma. Multi-marker association results are consistent with a highly polygenic contribution to asthma risk, including loci with weak effects that might be shared with other immune-related diseases, such as NDFIP1, HLA-B, LPP, and BACH2. INTERPRETATION The IL6R association further supports the hypothesis that cytokine signalling dysregulation affects asthma risk, and raises the possibility that an IL6R antagonist (tocilizumab) may be effective to treat the disease, perhaps in a genotype-dependent manner. Results for the 11q13.5 locus suggest that it directly increases the risk of allergic sensitisation which, in turn, increases the risk of subsequent development of asthma. Larger or more functionally focused studies are needed to characterise the many loci with modest effects that remain to be identified for asthma. FUNDING National Health and Medical Research Council of Australia. A full list of funding sources is provided in the webappendix.

Association of polymorphisms within the tumour necrosis factor (TNF) genes and childhood asthma

Tumour necrosis factor alpha (TNFα) is a potent modulator of immune and inflammatory responses, and has been implicated in a variety of autoimmune diseases, including asthma. Increased levels of TNFα have been detected in both sputa and bronchoalveolar lavage fluid of asthmatic subjects during acute attacks. Interindividual variation in TNFα levels may be genetically determined and polymorphisms within the TNF genes and nearby HLA Class II region have been associated with differences in TNFα production. Objective To investigate the association of differences in asthma‐related phenotypes with two biallelic polymorphisms: a G to A substitution at position −308 of the TNFα gene promoter (TNF1 and TNF2 alleles) and an NcoI polymorphism in the first intron of the lymphotoxin alpha gene (LT‐α*1 and LT‐α*2 alleles).

A POLYMORPHISM OF THE CC16 GENE IS ASSOCIATED WITH AN INCREASED RISK OF ASTHMA

Several quantitative traits associated with the asthma phenotype have been linked to markers on chromosome 11q13, although the gene responsible has yet to be well established. The gene for Clara cell secretory protein (CC16) is an ideal candidate for involvement in an inherited predisposition to asthma because of its chromosomal location, the role of the CC16 protein in controlling airway inflammation, and differences in levels of the protein between asthmatics and healthy controls. All three CC16 exons were screened in an unselected population of 266 subjects from 76 families and a cohort of 52 severely asthmatic children. A combination of single strand conformational polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, and restriction digestion was used. Mutation detection methods identified an adenine to guanine substitution in the CC16 gene at position 38 (A38G) downstream from the transcription initiation site within the non-coding region of exon 1. In the unselected population, 43.6% were homozygous for the polymorphic sequence (38GG) and 46.2% were heterozygous (38AG). All the asthmatic and unaffected children from both populations were selected for an unmatched case control analysis consisting of 67 asthmatic and 46 unaffected subjects. Those homozygous for the published sequence (38AA) had a 6.9-fold increased risk of developing asthma (p=0.049) and heterozygotes (38AG) a 4.2-fold increased risk (p=0.028). Modelling of genotype as a continuous covariate indicated evidence of a significant linear trend across the three genotypes (odds ratio=2.84 per unit increase in genotype code, p=0.018). These associations were independent of age, gender, and tobacco smoke exposure. These data and the known anti-inflammatory role of CC16 in the respiratory tract suggest that alteration to the gene at position 38 may contribute to asthma.

Polymorphisms in the β2‐adrenoreceptor gene are associated with decreased airway responsiveness

There are a number of candidate genes thought to play a role in the development of asthma. Polymorphisms at amino acid positions 16 (arginine to glycine) and 27 (glutamine to glutamic acid) of the β2‐adrenoreceptor (B2AR) gene are known to be functionally relevant and have been associated with more severe forms of asthma, nocturnal asthma and decreased airway responsiveness in asthmatic subjects.

Investigations into the role of ST2 in acute asthma in children

The ST2 gene is a member of the interleukin-1 receptor family and is located on chromosome 2q12, an area of the genome that has been associated with asthma. The soluble product of the ST2 gene, serum ST2 (sST2), has previously been shown to be elevated in adult asthmatic patients. This study investigated the potential role of ST2 in children with acute asthma. Children aged 2-16 years (n = 186) were recruited on presentation with acute asthma in the emergency department. Blood was obtained on presentation and during convalescence. Variables assessed included sST2 levels, a comprehensive assembly of clinical parameters and two polymorphisms in the ST2 gene, -26999G/A, located in the distal promoter region, and ala78glu polymorphism, on exon 3. The A allele of the -26999G/A polymorphism occurred more frequently in asthmatics compared with an unselected control group (P = 0.031). Serum ST2 levels were substantially higher during acute asthma compared with levels after the attack: 0.29 ng/ml (95% confidence interval: 0.23-0.36) and 0.14 ng/ml (0.12-0.17), respectively (P = 0.001) and were inversely related to eosinophil counts during an acute asthma attack (P = 0.002). The -26999AA genotype, as well as the AC haplotype, was associated with asthma severity scores (P = 0.05 and 0.02) compared with the -26999GA and GG genotypes. Serum ST2 levels were not associated with any of the studied genotypes or haplotypes. The observed associations of ST2 genotypes and haplotypes with acute asthma and asthma severity scores as well as the phenotypic differences associated with ST2 polymorphisms suggest that ST2 may play a role in the pathophysiology of asthma.

Association of polymorphisms in the β2-adrenoreceptor gene with higher levels of parasitic infection

Abstract The diminishing incidence of parasitic infection in westernised societies has been suggested to result in an increased prevalance of asthma. Asthma is a polygenic disease and genome screens have shown that genes on chromosome 5q31–33 are strongly linked to the disease. The gene for the β2-adrenoreceptor is located in this region and two polymorphisms have been identified that result in amino acid changes at positions 16 (ArgGly) and 27 (GlnGlu). To determine whether these polymorphisms influence asthma and parasitic infection, a genotype/phenotype study has been performed on a cohort of 126 children from Coche Island in Venezuela. There is a high incidence of asthma on the island and intestinal helminthiasis is endemic. Genotyping for both polymorphisms was carried out by using the polymerase chain reaction and allele-specific oligonucleotide hybridisation. Genotype frequencies in this cohort were consistent with other studies and both polymorphisms were in significant linkage disequilibrium. Individuals who were homozygous for Arg16 had significantly higher levels of specific IgE to Ascaris lumbricoides (P=0.002), significantly higher A. lumbricoides egg counts (P=0.001) and significantly larger wheal sizes following skin-prick testing with A. lumbricoides allergen (P=0.008). There was no association between either polymorphism and total serum IgE or asthma in this population. A combination of mast cell degranulation and the lung migratory phase of A. lumbricoides larvae may result in bronchoconstriction in infected individuals. These results suggest that the Gly 16 allele confers resistance to high levels of parasitic infection in this population. An alternative explanation for the association is that it may be the result of linkage disequilibrium with other genes in the chromosome 5q31–33 region.

Mutation screening of interferon‐gamma (IFNγ) as a candidate gene for asthma

Background Reduced levels of interferon gamma (IFNγ) mRNA and protein have been detected in the bronchoalveolar lavage fluid of atopic asthmatics. IFNγ is secreted by TH1 cells while IL‐4 and IL‐5 are secreted by TH2 cells and an imbalance in the TH1/TH2 response may be responsible for atopic asthma. The gene for IFN4gM is located on chromosome 12; a region of the genome which has been shown in linkage studies to be associated with asthma.

Opposite gene by environment interactions in Karelia for CD14 and CC16 single nucleotide polymorphisms and allergy

Background:  Finnish Karelians have a higher prevalence of allergic disease than Russian Karelians. As both populations are generally from the same ethnic group, the Karelian population offers a unique opportunity to analyse genetic and allergic disease interactions between ‘Western’ and ‘Eastern’ environments.

Anti-bacterial IgE in the antibody responses of house dust mite allergic children convalescent from asthma exacerbation

Background Atopic sensitization to the house dust mite (HDM) is associated with altered antibody responses to the nasopharyngeal colonizing bacterium Haemophilus influenzae and children admitted to the emergency department for asthma exacerbation have reduced IgG responses to HDM allergens.

In Utero Smoke Exposure and Role of Maternal and Infant Glutathione S-Transferase Genes on Airway Responsiveness and Lung Function in Infancy

RATIONALE Xenobiotics in the maternal circulation are capable of crossing the placental barrier so a reduction in the mother and fetuss detoxification ability due to genetic variation in the glutathione S-transferases (GSTs) could expose the fetus to higher levels of toxins. OBJECTIVES To investigate the interactive effects of maternal smoking during pregnancy with maternal and infant GST genotypes on airway responsiveness (AR) and lung function in infancy. METHODS GSTT1, GSTP1 and GSTM1 were genotyped in infants and mothers, in utero smoke exposure was evaluated by questionnaire, AR was assessed by histamine challenge and Vmax(FRC) was measured using the rapid thoracoabdominal compression technique. We investigated the interactive effects of maternal smoking during pregnancy with maternal and infant GST genes on AR and lung function at 1, 6, and 12 months and longitudinally throughout the first year. MEASUREMENTS AND MAIN RESULTS Infant and/or maternal GSTT1 nonnull was associated with reduced AR at 12 months and throughout the first year and increased Vmax(FRC) at 6 months. Maternal GSTP1 Val/Val or Ile/Val was associated with increased Vmax(FRC) at 6 months. In infants exposed to in utero smoke, infant and/or maternal GSTT1 nonnull was associated with reduced AR at 1 month and throughout the first year and increased Vmax(FRC) throughout the first year. Maternal GSTP1 Val/Val or Ile/Val was associated with increased Vmax(FRC) at 6 months. CONCLUSIONS GST genes may be especially important during fetal development as they may modify, through proficient detoxification, the effects of in utero maternal smoke exposure on AR and lung function in infants.