Using both a population-based and family-based approach, we have a nonsynonymous SNP, rs2272094, associated with CRTAM
that was replicated in an independent population. The risk of asthma exacerbation in patients homozygous in the minor allele of rs2272094 for low versus high vitamin D levels was an OR of 7.5 (95% CI, 0.93–60.43) in the CAMP population and 3.52 (95% CI, 0.69–18.05) in the Costa Rican replication population, with an overall OR of 4.4 (95% CI, 1.19–16.58) and a combined attributable risk of 56%. Genome-wide searches for gene-environment interactions, including those involving asthma,26
have been largely unsuccessful, and to our knowledge, none have been successfully replicated. That our result was consistent in both a population-based approach and a family-based approach and was replicated in an independent population for an interaction SNP suggests that this is a functional and causal SNP.
Viral infections are a major cause of asthma exacerbations,27
and vitamin D deficiency has been shown to be associated with increased susceptibility to viral respiratory tract infections.28
Therefore prevention of viral infections might be one mechanism for vitamin D’s involvement. Numbers of activated CD8+
T cells in peripheral blood are increased during viral respiratory tract infections,29
whereas NK activity is enhanced in acute asthma exacerbations.30
This implies a link between CD8+
and NKT cells and asthma exacerbation. Because vitamin D is known to up-regulate proteins that have antiviral properties,31
have an inhibitory effect on CD8+
T cell–mediated cytotoxicity,11
and are required for the normal function of NKT cells,12
it is possible that one mechanism by which vitamin D prevents asthma exacerbations is through CD8+
and NKT cells during viral infections.
expression is restricted to class I MHC T cells, including CD8+
and NKT cells. It is also one of the most highly expressed surface markers of activated human CD8+
and NKT cells.32
binds to Necl-2 on antigen-presenting cells and has been shown to promote cytotoxicity of NKT cells and IFN-γ and IL-22 secretion of CD8+
It has also been shown to regulate the immune response through the retention of CD8+
T cells within lymph nodes.34
Moreover, the lungs are one of the tissues with the highest expression of CRTAM
and it is thus plausible that variants in CRTAM
interact with vitamin D on asthma exacerbations, perhaps through prevention of viral illnesses. It also suggests a mechanism by which CD8+
T and NKT cells contribute to asthma exacerbation through IL-22 and IFN-γ.
There are a number of potential ways in which rs2272094 might meaningfully affect CRTAM function and expression in the context of altered vitamin D levels. Using the Eukaryotic Linear Motif resource for motif identification, we found that the rs2272094 change in amino acid from K to R corresponds to a change in motif from the KEN box to a proprotein convertase cleavage site, thereby potentially promoting the stabilization of the CRTAM protein.
Second, rs2272094 corresponds to the first base in the consensus sequence for binding of steroidogenic factor-1 (5′-AA GGTCA-3′; see Fig E3 in this article’s Online Repository
), which has been shown to play a role in ste-roidogenesis.35
Steroidogenic factor-1 is an orphan steroid receptor shown to regulate the transcription of P450 steroid hydroxylase genes.35
Because members of the cytochrome P450 superfamily are key enzymes in vitamin D metabolism,36
this suggests a possible mechanism by which CRTAM
might be linked with vitamin D metabolism. In addition, rs2272094 is part of a TTS, 5′-GGAAGAAAGG-3′. TTSs have the largest concentration in regulatory regions and are thought to contribute to gene expression.37
Triplex formation can lead to modulation in gene expression, transcription, protein binding, and targeting for DNA damage. A variant in this region might therefore have a number of functional consequences.
Finally, the minor allele in rs2272094 results in a change in an exonic splicing enhancer-binding site, AGAGAG, to one that is not, AGAAAG, which could prevent an entire exon from being transcribed.
Although it was the SNPs associated with CRTAM that replicated with nominal significance, it is interesting to note that 11 of the 23 SNPs examined were associated with the phospholipase C–like 1 gene (PLCL1). PLCL1 did not replicate in the Costa Ri-can cohort, however. Nevertheless, it would be interesting to examine PLCL1 in future replication studies.
One limitation in this current analysis is the slight difference in the phenotype in the 2 populations. In CAMP the phenotype is severe exacerbations resulting in either emergency department visits or hospitalizations in the year after entry into the trial. In contrast, in the Costa Rican study the phenotype is either emergency department visits or hospitalizations in the past year. To the extent that prior exacerbations predict future exacerbations,38
we believe that this limitation does not invalidate our replication results.
Another limitation is our moderate sample size. The small effects of gene-environment interactions are difficult to detect and even more difficult to replicate. Although a small number of studies have demonstrated gene-environment interaction through candidate SNP studies, only a few have done so through a genome-wide search,39–41
and to our knowledge, none have been replicated. Although we were able to achieve nominal significance and replication, a larger sample size in future studies will enable us to achieve genome-wide significance.
In summary, our genome-wide environment interaction study has identified a nonsynonymous variant in CRTAM, rs2272094, which interacts with circulating vitamin D levels in asthma exacerbation in children. The functionality of this variant is supported by gene expression studies and motif and sequence analyses. The association of CRTAM with CD8+ and NKT cells suggests biologically plausible mechanisms behind the role of these cells and vitamin D on asthma exacerbations in association with viral infections. These findings suggest a means by which asthma exacerbations might be prognosticated based on genotype and vitamin D levels, as well as a potential therapeutic intervention in a subset of the genotypically high-risk population.