A major challenge for candidate gene association studies is the selection of biologically relevant genes for evaluation. Our unique approach utilized multiple lines of evidence to select relevant candidate genes (). First, as nasal epithelial cells have been previously shown to be a good surrogate for bronchial epithelial cells 
, we took advantage of expression microarray data from these cells to identify genes differentially expressed in asthmatics and non-asthmatic controls. Next, using HapMap, we compared the allelic variation across each of these genes in populations with marked differences in asthma prevalence (CEU vs. CHB), reviewed the published literature to identify specific genes having potential application in the lung and genotyped tagging SNPs across these genes. Using this approach, we determined that SNPs within six genes were associated with asthma (). Many of these associations were also observed in one or more of our 4 independent populations either at the SNP or gene level ( and ), and the association with KIF3A
was associated in three of four populations examined in addition to our discovery population. The Puerto Rican and Mexican GALA trios not only allowed us to evaluate our results across racial/ethnic groups, but to evaluate genetic associations in populations with potentially different environmental exposures.
For candidate gene selection, researchers have used gene expression results 
, FST 
, and biologic relevance as gleaned from the literature 
, but no study has combined these strategies. We found that the combination of approaches for candidate gene selection was superior to using one type of data. Indeed, in an independent screen of SNPs in genes found to have dysregulated epithelial gene expression in uncontrolled asthmatics, we found that SNPs with high FST
values were more likely to be associated with asthma than those with low FST
values, including the six genes evaluated in this study, supporting the validity of our gene selection approach. Further, when we randomly selected genes from those that were differentially expressed, we found our selection method was statistically superior. These data point to a strong relationship between epithelial cells and asthma and substantiate the validity of this approach to identify genetic biomarkers of complex disease. Collectively, our approach may be applicable to other complex diseases that show varying prevalences across human populations, and may be a useful tool to select novel gene candidates for assessment of disease associations in other pertinent cell types and tissues.
Using this approach, we have identified KIF3A
as a possible susceptibility gene for childhood asthma and allergic disease. We found that seven of nine KIF3A
SNPs genotyped were significantly associated with asthma in our discovery Caucasian population and in independent Caucasian and African American populations – all from Cincinnati (). SNPs in KIF3A
were also to a lesser degree, associated with other allergic diseases independent of asthma demonstrating the importance of appropriate phenotyping and selection of controls in genetic studies of asthma (). If we had merely compared asthmatics to non-asthmatics without determining the non-asthmatics allergic status, we might have missed the association with KIF3A
. Importantly, all of the disease associated KIF3A
SNPs and neither of the two non-associated KIF3A
SNPs displayed significant population differentiation in HapMap CEU and CHB populations (individual FST
values>0.25; ). The observed association was strongest for KIF3A
rs7737031 which had a PAR for asthma of >18%. Carriers of rs7737031 have more than double the odds of having asthma. Similar to the PAR of this KIF3A
variant, the PAR for myocardial infarction was 21% in individuals with the previously reported rs10757278 variant located in adjacent CDKN2A
. We also directly genotyped this SNP and evaluated its association with asthma in Puerto Rican and Mexican populations in the GALA Study. Puerto Ricans and Mexicans living in the US have higher and lower asthma prevalences, morbidity, and mortality rates than Caucasians, respectively. Interestingly, our analysis reveals that KIF3A
rs7737031 was significantly associated in Puerto Ricans and not Mexicans.
KIF3A SNP associations with allergic disease.
KIF3A is a heterotrimeric member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia and flagella 
among other roles. Cilia, together with mucus and the airway surface liquid layer, make up the mucociliary apparatus that clears inhaled allergens or other particles from the lung. Defective mucociliary clearance is a characteristic feature of several genetically linked airway diseases including asthma and cystic fibrosis 
, yet the mechanisms responsible for poor mucus and/or allergen clearance from the airways remain largely unknown.
is located on 5q31, immediately upstream of IL-4
, we also noted a strong LD between IL-4
SNPs in our discovery cohort, a finding that is consistent with another published report 
. Examination of International HapMap data 
indicates that this LD exists across many other populations as well making it difficult to determine if one or both genes confer risk. Numerous studies have reported associations with IL-4
, asthma and other allergic diseases 
. It is possible that some of the previous asthma associations reported between asthma and IL-4
may reflect the LD with KIF3A
. Therefore, we further examined the biologic plausibility of KIF3A
as an asthma susceptibility gene by examining gene expression in the lungs of mice. We found expression of KIF3A
was significantly reduced in HDM-treated mice compared to controls (). We speculate that in asthmatics specifically, diminished KIF3A
expression might be important in allowing the lung to repair itself after exacerbation. Alternatively, diminished KIF3A
expression may contribute to the lungs' inability to clear mucus and remove inhaled particles and aeroallergens therefore exacerbating the asthma and/or allergic phenotypes. Individuals with polymorphic cilia genes may have further reduced cilia gene expression, diminished ciliary function and increased allergen exposure resulting in even greater susceptibility to asthma and allergic disease. Regardless of the mechanism of action, KIF3A
is clearly down-regulated in asthma, supporting a role for this novel gene in the pathogenesis of this disease. While our analyses of tagging SNPs provide strong evidence of a gene-disease association, it will be important to investigate the combined effects of IL-4
in future studies and to identify specific causal variants in KIF3A
.These findings emphasize the importance of evaluating multiple genes and critically exploring the biological relevance of genes previously unknown to influence disease susceptibility.
In summary, our study took advantage of our previously published evaluation of nasal epithelial cell-derived RNA from asthmatic and non-allergic children 
, population differences in asthma prevalence, tagging SNPs in the HapMap database, and the published literature to identify six genes (ADCY2
) for detailed and targeted genetic testing. The most strongly associated gene, KIF3A
, was first reported by our group as having a role in asthma in 2009 
. KIF3A has also been associated with aspirin sensitive asthma 
. Here, we verify that KIF3A
is a novel candidate gene for childhood asthma and show that its expression is down regulated in nasal epithelial cells in asthmatics. Our success supports the validity of our approach for identifying asthma candidate genes with a high likelihood of exhibiting association in specific variants. As the level of genomic data continues to increase, it will be imperative to develop methods which can help focus studies. By demonstrating that we can identify associations using this approach and that these associations can be replicated, we have provided a novel framework for the identification of candidate genes.