This is the first study to report gene-based replicated associations between the CRHR2
gene and acute bronchodilator response upon short-acting β2 agonist administration in asthmatic subjects. In our primary and two replication cohorts, different SNPs of CRHR2
were found to be associated with acute bronchodilator response to albuterol, however, the physical proximity of these SNPs with each other (between exon β1a and exon 3) suggests that the causative variant(s) may reside towards the 5’ end of the gene. By comparing the haplotypes showing the most significant associations in each cohort, the associated region spans approximately 8.7kb between introns β1b and 2 (). The haplotype analyses detected the common haplotypes (frequency between 60% - 78%) to be associated with acute bronchodilator response in the three cohorts. The haplotypes are of different length, yet in CAMP and LODO, allele rs7793837A resides in almost all the associated common haplotypes. In Sepracor, the associated haplotype with a frequency of 78% consisted of 2 neighboring SNPs (rs4723002 and rs1003929) 1 and 7 kb downstream of variant rs7793837. We speculated that variant rs7793837 was associated in Sepracor because (1) it is not the causative variant but in LD with the causative variant, and (2) Sepracor is phenotypically different from CAMP and LODO. This cohort has a higher mean bronchodilator response due to the recruitment requirement of subjects with >15% bronchodilator response (Sepracor: -40.98% (standard deviation of 20.98), CAMP:10.75 (sd 10.13) and LODO: 9.67 (sd 11.07)). A different causative variant, also residing at 5’ end of CRHR2 may be responsible for the association observed in Sepracor. This 5’ region encodes the N-terminal extracellular domain of CRHR2, where interactions with ligands occur (reviewed in [19
]). In addition to encoding the N-terminal, this region houses multiple exons 1 (β1b, γ1 and α1), where various isoforms of CRHR2 are translated depending on the splicing transcripts being translated [19
]. Additional sequencing of the 5’ end of CRHR2
was carried out to identify the causative variant(s). Approximately 32 kb of genomic regions (exons β1a, β1b, γ1, α1, 2 and flanking intronic regions) were sequenced in 48 CAMP subjects, revealing 3 novel intronic variants located 199bp upstream and 26 and 74bp downstream of exon β1a, respectively. Two of the 3 novel variants were chosen for their potential functions and were genotyped in a subset of CAMP subjects, but no association was observed with acute bronchodilator response (data not shown). Although the original 28 SNPs panel did not cover exon β1a, subsequent sequencing and genotyping efforts ensured exon β1a and surrounding intronic regions were investigated.
Haplotypes demonstrating the most significant association with acute bronchodilator response in CAMP, SEPRACOR and LODO
The rationale for testing for associations in both pediatric and childhood asthma cohorts is to establish generalization of the associations between CRHR2 genetic variants and acute bronchodilator response across different age groups; to determine whether the association was a developmental, an aging, or both a developmental and aging phenomenon in the lungs. Our findings that different SNPs were associated with acute bronchodilator response in the three cohorts and that these SNPs reside at the 5’ end of CRHR2 suggest that the associations observed are likely due to linkage disequilibrium between associated SNPs and the true phenotype causing variant(s), in both childhood and adult asthma.
The 5’ end of CRHR2
is of great biological interest. The CRHR2 protein has 3 isoforms (CRHR2α, CRHR2β, and CRHR2γ) which differ at the N termini, and are encoded by the 5’ end exons of CRHR2
. Translations of exon β1a and exon β1b encode the N terminus of the CRHR2β isoform, and exon 1α and 1γ encode the N termini of CRHR2α and CRHR2γ, respectively [18
]. In vitro
study has demonstrated that the three isoforms have different downstream signaling capacities [18
]. We speculate that the causative variant(s) may influence acute bronchodilator response through regulating translations of CRHR2
into various isoforms.
How genetic variations in CRHR2
function contribute to differential responses to β2-agonist remains to be investigated. Literature has presented complex interactions between β2 AR and other signaling pathways, based on which we propose three molecular mechanisms that could potentially explain the association of CRHR2
variants with bronchodilator response. First, CRHR2 signaling may desensitize β2 AR function in asthmatics. β2 AR signaling can be desensitized not only by its own activation but also by signaling through other G protein-coupled receptors. This cross-talk, which is referred to as heterologous desensitization (versus homologous desensitization by its own signaling), has been observed between β2 AR and PGE2 receptors [30
]. This mechanism predicts that individuals with higher CRHR2 signaling capacity may have reduced bronchodilator response due to stronger cross-desensitization. A second mechanism takes into consideration the documented anti-inflammatory role of CRHR2 [12
]. In this case, reduced CRHR2 function may lead to increased production of pro-inflammatory cytokines, many of which will down-regulate the signaling through β2 ARs [31
]. The last mechanism concerns the possible synergistic interaction between CRHR2 and β2 AR since both of them relax smooth muscles by stimulating cAMP production. If synergistic contribution from CRHR2 is pivotal for β2 agonists to achieve effective bronchoprotection in asthmatics, patients carrying CRHR2 alleles that have lower biological activities will have dampened therapeutic response to β2 agonists. Synergy between G-protein coupled receptors (GPCRs) has been demonstrated in many biological systems where activation of one GPCR can amplify the signaling events in a parallel but separate pathway [32
]. To determine which one of these three proposed paradigms is valid, we need to perform physiological studies on mice that were engineered to be CRHR2-deficient as well as biochemical analysis of signaling interaction between CRHR2 and β2 AR in primary airway smooth muscle cells.
This study has limitations. First, we did not detect a single SNP that was associated across the 3 cohorts. Second, the significant associations detected would not reach significance level when corrected for multiple testing. However, in spite of these limitations, we felt that by detecting associations with SNPs covering a region within the gene across 3 populations, the findings that CRHR2, as a gene, is associated with acute bronchodilator response is likely to be valid. The lack of any single SNP being associated in all three cohorts suggests that the genetic effect of CRHR2, although present, is likely to be weak. Being a complex trait, the number of genetic variants explaining inter-individual variations in bronchodilator response is likely to be high, with each variant exerts low to moderate effect. Our findings suggests that genetic variants of CRHR2, in addition to the widely studied β2AR and many yet to be characterized genetic variants, influence acute bronchodilator response to short acting β2A. Despite our subsequent sequencing and genotyping effort of exons at the 5’ end of CRHR2, we did not detect any causative variants, hence, the causative variants are likely to be in untranslated regions. Additional exploration of the region will be needed to identify the causative variants affecting acute bronchodilator response so that subsequent screening tests can be developed to predict in advance the efficacy of administering β2 agonists as an asthma treatment in asthmatic individuals.