The gene encoding ADisintegrin And Metalloprotease (ADAM) 33 was the first asthma susceptibility gene to be discovered by positional cloning.¹ In 460 families enriched with asthma, linkage analysis using microsatellite markers spaced ∼9 cM apart revealed a region on chromosome 20p13 that carried one or more asthma genes, achieving a Maximum Lod Score (MLS) of 2.24 at 9.99 cM. The addition of further markers at 1.2 cM increased the MLS to 2.94 at 12.1 cM which further rose to 3.93 when bronchial hyperresponsiveness was included in the definition of asthma despite halving the sample size, thereby exceeding the threshold for genome wide significance. Physical mapping, direct cDNA selection, and sequencing of DNA cloned into bacterial artificial chromosomes (BACs) identified 25 candidate genes. Linkage disequilibrium mapping of single nucleotide polymorphisms (SNPs) on 23 genes spanning the peak of linkage together with case-control and family based association analyses revealed that ADAM33 accounted for the linkage signal.

Several features of this initial report raised questions regarding the generalisability of the results.² Firstly, although significant evidence for linkage was observed, this region on chromosome 20p had not been identified in previous genome wide screens in asthma. Secondly, the initial publication did not have a truly independent replication sample. Thirdly, no single SNP demonstrated significant association in both the UK and US populations that made up the total sample when these were analysed separately. Finally, no functional data regarding the role of associated SNPs in alteration of gene expression and/or function and in the development of asthma phenotypes were presented.

Since 2002 there have been a number of separate replication studies in diverse ethnic populations. The first by Howard et al³ examined eight SNPs in the 3′ portion of ADAM33 reported in the original study to be associated with asthma in four unique asthma populations comprising African American, US white, US Hispanic, and Dutch white populations. Significant associations with at least one SNP and asthma were found in each of the populations (p = 0.0009–0.04) with multiple SNPs associating with asthma or its partial phenotypes in some of the populations. Further replication has been reported in separate case-control and family based association studies in Germany,⁴ Korea,⁵ and Japan.⁶ However, there are two published studies showing no evidence of association⁷ or weak association.⁸

It is therefore timely that, in this issue of Thorax, Blakey and colleagues report the result of a meta-analysis involving eight separate populations totalling 1299 cases and 1665 controls used in case-control association analysis and 4561 families used in transmission disequilibrium tests (TDTs).⁹ In both types of analysis several SNPs were significantly associated with asthma. The important point the authors made is that, based on allele frequencies for the ST+7 G allele of 84.9% in the asthmatic population and 79.1% in the controls and an asthma prevalence of 8%, this SNP would potentially contribute to ∼50 000 excess asthma cases in the UK population.

What is important to point out is that most case-control and family based association studies looking for disease related genes in complex disorders are statistically underpowered and that far larger sample sizes are needed. Part of the reason for this is the existence of genetic heterogeneity with any one gene varying in its influence over a disease phenotype between populations with differing genetic backgrounds and differing environmental exposures.^10,11 For example, natural selection may have acted on a disease gene haplotype differentially in different populations as recently described for the IL-4 locus on chromosome 5q31–34.¹² Another example is the NOD2/CARD 15 gene on chromosome 16 which has been associated with Crohn’s disease in some but not all populations.¹³ Because a statistical association is not revealed in a particular population irrespective of size does not necessarily mean that the gene in question is not contributing to the phenotype, but the mode of its influence may be complex involving gene-gene or gene-environmental interactions.¹⁴ However, as the number of independent studies increases, it would be valuable to accrue the evidence systematically as was reported for linkage analysis for asthma on chromosome 5 (the Consortium on Asthma Genetics).¹⁵ With the recent establishment of the Network of Excellence for Asthma and Allergy (GA²LEN), there is a unique opportunity to further develop meta-analyses for candidates such as ADAM33. The study by Blakey et al is an excellent example of the power of this approach.

At present the SNPs that cause the dysfunction in ADAM33 predisposing to asthma are not yet clear, although several SNPs (T1, F+1 and ST+7) are coming through in a number of different studies as being associated with asthma. However, because of the very large degree of linkage disequilibrium between many SNPs in ADAM33 so far identified (possibly in excess of 100), genetics alone is unlikely to cast much further light on the disease related variants although new analytical methodologies are being developed.¹⁶ Some clues about how ADAM33 may influence the asthma phenotype are emerging. In 200 Dutch patients with asthma who had regular lung function measurements made over 20 years, the rare alleles of the SNPs S-2, T-1 and T-2 of the ADAM33 gene were associated with a significant excess decline in baseline forced expiratory volume in 1 second (FEV₁) of 23.7–30 ml/year.¹⁷ These data imply a role for ADAM33 in airway wall remodelling which is known to contribute to chronic airflow obstruction in moderate to severe asthma.¹⁸ A second study conducted in infants born of allergic/asthmatic parents in Northern England (^NACMAAS) has revealed positive associations between SNPs of ADAM33 and increased airway resistance measured by plethysmography at age 3 and again at age 5 years, with the strongest effect seen in the homozygotes.^19,20 These data support the idea that alterations in the expression or function of ADAM33 is in some way involved in impairing lung function in early life and, as a consequence, increasing the risk of asthma developing.

The initial study¹ as well as others^3,5 have revealed some of the strongest associations when bronchial hyperresponsiveness (BHR) is incorporated into the asthma phenotype. The cellular provenance of ADAM33 mRNA and protein in being restricted to mesenchymal cell types (fibroblasts, myofibroblasts and smooth muscle) reinforces the view that this molecule is involved in the pathophysiology of BHR and airway remodelling rather than the immunological or inflammatory components of asthma.^1,21 Expression of full length ADAM33 in mammalian cell lines has shown that the metalloprotease domain of ADAM33 is functional^22,23 but the biological targets of the metalloprotease activity are as yet unknown. In cell based sheddase assays ADAM33 functioned as a negative regulator of β-amyloid precursor peptide (APP) cleavage and mediated some constitutive shedding of stem cell factor (SCF, ckit ligand); however, the kinetics of these cleavage reactions would indicate that these two proteins are not the natural substrates.²³

Six alternatively spliced variants of ADAM33 in airway fibroblasts have recently been described including one putative secreted variant.²⁴ Ninety percent of ADAM33 mRNA is retained in the nucleus and subtle differences in the composition of nuclear and cytoplasmic mRNA indicate important events in both splicing and selecting of ADAM33 transcripts for processing into proteins. What is of great interest is that none of the six variants contain the metalloprotease catalytic domain, suggesting possible other key functions of the molecule—for example, in cell fusion and adhesion.²⁵

There is still much to find out about this fascinating and complex molecule in relation to the development and progression of asthma. Added to it are three further new asthma/allergy genes identified by positional cloning: PDH Finger Protein II (PHF11) on chromosome 13q14 which encodes NY-REN-34, a protein first described in patients with renal cell carcinoma;²⁶ dipeptidyl diptidase 10 (DDP10) on chromosome 2q14;²⁷ and G protein-coupled receptor for asthma susceptibility (GPRA) on chromosome 7p.²⁸ For each of these genes, as for ADAM 33, determining normal functions and how these are disordered in asthma related alleles is the real future challenge. We are now entering the new research era of translational science and the rebirth of experimental medicine as a research focus.²⁹