This study identified PCDH1
as a novel gene for BHR in children and adults. Interestingly, we have provided evidence that PCDH1
, particularly rs3797054, is important in the development of BHR in a sample of families with asthma. In addition, PCDH1
is associated with BHR in two population-based samples from the Netherlands and the United States, irrespective of the presence of asthma. Moreover, we present strict replication of a second gene variant in PCDH1
, a 3-bp insertion-deletion, in three additional study samples ascertained for asthma. Finally, loose replication (28
) was observed in the Tucson cohort and a UK family study for a coding SNP not associated with BHR in the primary Dutch population. Of importance for the relevance of the PCDH1
gene for BHR and asthma, PCHD1 mRNA and protein expression was shown in macrophages and airway epithelial cells from subjects with asthma and control subjects.
To interpret these findings, several strengths and limitations need to be considered. First, to our knowledge this is the first gene that is identified by positional cloning for an intermediate phenotype of asthma, BHR. PCDH1
gene variants are associated with BHR in families with asthma as well as two general populations not ascertained for asthma. Previous epidemiological data have shown that BHR is a risk factor for asthma, even in subjects without respiratory symptoms (4
). Based on these data, we hypothesize that mechanisms related to the function of PCDH1
contribute to susceptibility to BHR and subsequent asthma development. This may implicate that PCDH1 dysfunction plays an early role in asthma pathogenesis.
Second, in all study populations, a direct agonist of BHR was used (either histamine or methacholine). Our findings suggest that PCDH1 function is not specific for BHR induced by either methacholine or histamine. Previous work has indicated that BHR to histamine and methacholine is highly correlated (29
). We suggest that further work should be performed on the role of PCDH1
in indirect measures of BHR, such as exercise or adenosine-monophosphate.
Third, we used the positional cloning approach that has been previously used to identify DPP10
). This approach is based on identification of a genetic association with a microsatellite marker used for linkage analyses, to subsequently fine map the region that is in possible linkage disequilibrium (LD) with this marker and the gene of interest. Our fine mapping results indicate that we have adequately screened the region for SNPs in other genes that may be in LD with PCDH1
, given the extent of LD of about 100 kb in this region in the Dutch population ().
Fourth, we identified PCDH1
in a primary family population, in which linkage to BHR was reported (6
). We did not correct for multiple testing, but rather performed extensive replication studies of significant and/or functional SNPs in seven independent populations. We interpret the consistent signal in parents and offspring in the Dutch families as internal validation of this genetic association in the Dutch families. Moreover, the strict replication of two PCDH1
gene variants with the same risk allele being associated with BHR in the same direction in four independent populations provides strong support for a role of PCDH1
in BHR (28
). Specifically, we found strict replication with regard to phenotype and genotype for Ala750Ala and IVs3_116. Loose replication with regard to genotype was observed for Ala514Thr. The reasons for this allelic heterogeneity are yet to be determined, but include the presence of multiple functional SNPs, other SNPs that are in linkage disequilibrium with the associated SNPs, or gene by environment interaction. Because gene variants on 5q31-q33 have been shown to interact with ETS exposure in utero/early life, we investigated the association of PCDH1
with BHR in ETS exposed and nonexposed children in four populations. These gene–environmental interaction analyses (Table E3) in the study cohorts revealed evidence of gene–environment interaction for PCHD1
in the UK family cohort only, but not in the other populations. However, the power to detect such an interaction was low.
We suggest characterizing the functional role of these gene variants in PCDH1. Ala750Ala and IVS3_116 are localized in the 3′UTR of exon 3 and may affect mRNA stability or splicing, whereas Ala514Thr is localized in the fifth cadherin repeat of the extracellular domain and may affect cell–cell adhesion.
The Protocadherin 1
) has five exons and encodes multiple mRNA isoforms through alternative splicing (Table E5). There are two annotated isoforms: a three-exon isoform and a five-exon isoform. The three-exon isoform lacks the major part of the cytoplasmic domain, which encodes conserved signaling sequences CM1, CM2, and CM3 (22
). Protocadherins are believed to play an important role in homologous cell adhesion and organ development, in particular within the neural system (30
). Overexpression of PCDH1 induces calcium-dependent cell–cell adhesion and membrane expression of PCDH1 in a mouse fibroblast L cell assay (27
). Protocadherin 1, like PCDH, belongs to the δ1-protocadherin family of transmembrane proteins. δ1-Protocadherins are characterized by six or seven cadherin repeats in the extracellular region, and three conserved regions designated CM1, CM2, and CM3 in the intracellular domain (31
). The conserved region CM2 interacts with protein phosphatase 1α (PP1α) in PCDH7 (31
). Interestingly, PP1α plays an important role in lung development, as inhibition of PP1α in a mouse model led to impaired lung development and branching morphogenesis (32
). Finally, in a skin keratinocyte wounding model, PCDH1 mRNA was significantly up-regulated 24 hours after wounding, compatible with a role of PCDH1 in epithelial repair (33
The expression pattern of PCDH1 is consistent with expression in the apical adhesion complex of airway epithelial cells. We therefore hypothesize that PCDH1 plays a role in epithelial integrity of the airways and that loss of function of PCDH1 is associated with increased BHR, which may lead to symptomatic asthma (34
). It is tempting to speculate that PCDH1 dysfunction may provide a functional explanation for the observed epithelial vulnerability and increased epithelial shedding in asthma (35
). Further investigations will address the functional relevance of genetic variations in PCDH1
in epithelial cell adhesion and its interaction with environmental tobacco smoke exposure. Moreover, these finding may be relevant for other diseases in which the integrity of the epithelium is a potential pathogenetic mechanism, such as atopic dermatitis (36
) and celiac disease (37
). We therefore suggest performing genetic studies of PCDH1
in these diseases.
In conclusion, this is the first report of a gene specifically identified for HR, an important hallmark of asthma. Further investigations in PCDH1 function may provide novel insight into its role in the integrity of the airway epithelium in BHR and asthma development.