Using an interdisciplinary genomics approach we investigated vitamin D and lung outcomes. SGPP2, a phosphatase involved in the sphingosine-1-phosphate signaling pathway, was identified in all stages of the study as a promising candidate gene contributing to vitamin D-mediated associations with lung function. SGPP2 is differentially expressed in vivo in lung epithelial cells by serum 25(OH)D. eQTL analysis demonstrates that sequence variants in SGPP2 are associated with lung cell gene expression. Although the eQTL finding does not prove that vitamin D regulation affects gene expression, the location of associated variants in regulatory regions supports the hypothesis of vitamin D regulation. Furthermore, a group of 3 linked SNPs in the SGPP2 promoter region are associated with lower FEV1, a reduced FEV1/FVC ratio, and a 2–3 fold increased risk of airflow obstruction in African-Americans, suggesting that a causal variant in this region may affect SGPP2 function and/or vitamin D binding, and, consequently, lung outcomes. Additionally, a SNP in SGPP2 is associated with FEV1 in Health ABC European-Americans and SGPP2 variants were also associated with FEV1 in the Framingham Heart Study, confirming effects across racial groups and in two cohort studies. This multi-faceted approach identifies putative mechanistic pathways for observed vitamin D—lung function associations while reducing the chance of false positive results.
SGPP2 plays a key role in the sphingolipid signaling pathway through dephosphorylation of sphingosine-1-phosphate (S1P) to sphingosine, which is then converted to ceramide or back to sphingosine-1-phosphate by other enzymes . Sphingosine-1-phosphate acts as both an intracellular and extracellular signaling molecule, and regulates critical cell processes including apoptosis, cell growth, and immune function [25, 26]. Altered sphingolipid concentrations have important ramifications for lung function; ceramide concentrations are elevated in COPD, contributing to lung alveolar destruction . Little research exists on SGPP2, although a 2006 paper showed that SGPP2 is up-regulated in response to inflammatory stimuli in endothelial cells, suggesting a possible role in mediating inflammation in lung tissue . However, SGPP2’s biological function to alter sphingosine-1-phosphate concentrations suggests that this gene contributes to the regulation of sphingolipid signaling pathways in lung tissue.
We identified several additional genes, namely DAPK1, KCNS3, and FSTL1, and all three had mechanistic links to lung function identified through gene ontology analysis and literature reviews (Additional files 11 and 12). Expression of all three genes was strongly associated with serum 25(OH)D, and variants in these genes were associated with pulmonary function in the Health ABC cohort study. However, variants were not replicated in the Framingham Heart Study, nor were there observed eQTL associations. DAPK1, which is down-regulated by 1,25(OH)2D both in vivo and in vitro, is a pro-apoptotic kinase linked to cytoskeletal remodeling and regulation of inflammatory gene expression in macrophages [28, 29]. SNPs in KCNS3, which encodes a voltage-gated potassium channel protein, were associated with airway hyperresponsiveness in past studies , which is of interest given postulated associations of airways hyperresponsiveness with an accelerated rate of FEV1 decline and risk of COPD . FSTL1 up-regulates pro-inflammatory cytokines; in mice, the highest expression level is in lung . Dexamethasone, which is a glucocorticoid used to treat both asthma and COPD, is associated with expression of both KCNS3 and FSTL1; interestingly, there are striking similarities in the effects of dexamethasone and 1,25-dihydroxyvitamin on the expression of these genes. The combination of 1,25-dihydroxyvitamin D with dexamethasone was investigated in vitro as an anti-inflammatory treatment; our results suggest the strong possibility of synergistic effects for this treatment combination (Additional file 12 for references).
A major strength of this study is that it translates in vitro animal and cell culture studies to an in vivo study, and then extends to study population-level SNP associations with lung phenotypes, which are partially replicated in an independent cohort. The multi-stage approach identified SGPP2 as a promising vitamin D-responsive gene for further study. The demonstration of differential gene expression in lung tissue associated with the physiologic range of 25-hydroxyvitamin D in a diverse sample of free-living humans confirms in vitro studies, and, while our study does not manipulate vitamin D, the in vivo evidence of association is novel. The Health ABC population-based cohort study included high-quality spirometry, detailed information on confounding factors such as smoking and population stratification, and comprised 40% African-American participants, thus allowing consideration of this understudied population in genomic research. FEV1 is a predictor of all-cause mortality , and thus SNP—FEV1 associations are clinically relevant. Although associations between SNPs and the FEV1/FVC ratio were also investigated, the associations were not as strong as for FEV1. Thus, vitamin D may have a stronger association with overall lung health versus the risk of COPD. This study identifies plausible biological mechanisms that support a true effect of vitamin D on lung function, and will help to guide the design and analysis of randomized controlled intervention trials of the role of vitamin D in lung disease.
Given that the microarray analysis was used exclusively as a candidate screen, limitations including the lack of qPCR confirmation (not possible due to sample volume limitations), use of nominal P values, and the lack of race-stratified analysis (not possible due to sample size limitations) are less of a concern. As expected, the proportion of participants in the race/ethnicity groups varied by tertile of serum 25(OH)D given the role of skin pigmentation in vitamin D synthesis in response to sunlight . Race may either confound the serum 25(OH)D—gene expression association, or, race may be a causal antecedent variable that, in part, causes serum 25(OH)D concentration and, in turn, differences in gene expression; adjusting for race may be an over-adjustment. Of note, in regressions adjusted for race the regression coefficients for the serum 25(OH)D—gene expression association were similar to unadjusted analyses.
While the studies were all cross-sectional, which limits causal inference, the harmony of findings across different designs partly mitigates this concern. Although it would have been ideal to use the same samples in all studies (that is, expression, eQTL and SNP—lung function studies), practical limitations led to the use of different samples in each phase. Finally, although gene-level replication was observed for SGPP2 and DAPK1, the specific SNPs associated with FEV1 in Health ABC did not reach statistical significance in FHS. We hypothesize that the SGPP2 SNPs identified in the two cohort studies may be tagging the same unknown causal variant(s) or there may be multiple SGPP2 regulatory regions associated with lung function. Additionally, the strongest SNP—lung function associations in Health ABC were in African-Americans, and, because FHS includes only European Americans, the replication was partial. In summary, SNPs in SGPP2 were statistically significantly associated with lung outcomes after FDR multiple testing adjustment and a highly statistically significant lung eQTL was identified for SGPP2; SGPP2 emerged as a clear candidate in all stages of this work.