SNP-set analysis replicates acute lung injury genetic risk factors

Abstract

Background: We used a gene - based replication strategy to test the reproducibility of prior acute lung injury (ALI) candidate gene associations.

Methods: We phenotyped 474 patients from a prospective severe trauma cohort study for ALI. Genomic DNA from subjects' blood was genotyped using the IBC chip, a multiplex single nucleotide polymorphism (SNP) array. Results were filtered for 25 candidate genes selected using prespecified literature search criteria and present on the IBC platform. For each gene, we grouped SNPs according to haplotype blocks and tested the joint effect of all SNPs on susceptibility to ALI using the SNP-set kernel association test. Results were compared to single SNP analysis of the candidate SNPs. Analyses were separate for genetically determined ancestry (African or European).

Results: We identified 4 genes in African ancestry and 2 in European ancestry trauma subjects which replicated their associations with ALI. Ours is the first replication of IL6, IL10, IRAK3, and VEGFA associations in non-European populations with ALI. Only one gene - VEGFA - demonstrated association with ALI in both ancestries, with distinct haplotype blocks in each ancestry driving the association. We also report the association between trauma-associated ALI and NFKBIA in European ancestry subjects.

Conclusions: Prior ALI genetic associations are reproducible and replicate in a trauma cohort. Kernel - based SNP-set analysis is a more powerful method to detect ALI association than single SNP analysis, and thus may be more useful for replication testing. Further, gene-based replication can extend candidate gene associations to diverse ethnicities.

Figure 1

A: Study population. All trauma patients were screened. Eligible subjects were severely injured, with an injury severity score (ISS) ≥ 16, and were admitted to the intensive care unit (ICU). Exclusion criteria included isolated head injury and discharge or death within 24 h. Subjects with adequate DNA were genotyped with the IBC SNP array and were classified by genetic ancestry, African (AA) or European (EA), to create haplotypes for ALI candidate genes. B: Overview of study design and results. Large scale genotyping data was available for 474 subjects in a trauma cohort. A literature search using the terms “acute lung injury polymorphism,” “acute respiratory distress polymorphism,” or “genetic association lung injury,” limited to human species, was performed in May 2011 and returned 38 genes. Nine genes (IL1RN, ORMDL3, THMD, DARC, DIO2, PRKAG2, ISG15, VWF, and BVES) were cited in abstract form only, and were excluded due to lack of detail about the population or genetic variant studied. Of the 29 remaining genes (Table 2), 4 were not covered by the IBC platform. The remaining 25 genes underwent haplotype imputation for each ancestry and the SNP-set kernel association test was implemented for 3 kernel functions: linear, identical by state, and quadratic. Four genes in subjects of African ancestry and 2 genes in subjects of European ancestry replicated their association with ALI in this trauma cohort.

Figure 2

Linkage disequilibrium (LD) plots for African ancestry (AA) trauma subjects for 4 genes with positive ALI associations. Previously reported ALI-associated SNPs are circled in red, whereas the haplotype block detected to be ALI – associated by one or more kernel function is outlined in yellow. The grayscale reflects the pairwise LD as measured by r², with high LD in black and low/absent LD in white. The ALI – associated genes are (A) IL6, (B) IL10, (C) VEGFA, and (D) IRAK3.

Figure 3

LD plots for European ancestry (AA) trauma subjects for 2 genes with positive ALI associations. Plots for the genes VEGFA (A) and NFKBIA (B) are shown. Grayscale reflects the pairwise LD as measured by r², with high LD in black and low/absent LD in white. As in Figure 2, previously reported ALI – associated SNPs are circled (red) and the yellow lines outline the haplotypes detected by kernel function(s) as being associated with ALI. Whereas the block detected in VEGFA is defined by the previously reported SNP rs3025039, the association with NFKBIA does not overlap the previously reported NFKBIA ALI – associated locus.

Figure 4

Representative kernel matrix plot of the IL10 gene in AA subjects. For each matrix, the ALI or non-ALI population of haplotypes is plotted against itself according to the kernel function. Each cell on the matrix reflects the extent of haplotype sharing between ALI subjects or non-ALI subjects. The cell size reflects the frequency of the haplotype, such that common haploypes are larger rectangles. The color reflects the kernel function value for each haplotype among ALI or non-ALI; more intense red coloration reflects a higher degree of similarity between pairs of subjects based on their diploid genotypes. Comparing the top and bottom plots in each box, one observes that the ALI (top) plots are distinct for each kernel function, but the plots are most discordant for the quadratic function, which allows for 2-way interactions between SNPs within the haplotype block to inform the association. Accordingly, the p- value for the quadratic kernel test with ALI was the most statistically significant.

Figure 5

Representative kernel matrix plot of the VEGFA gene in EA subjects. As in Figure 4, the plot displays the frequency of haplotype sharing within ALI cases or non – cases, where size of the cell reflects the overall haplotype frequency and the red coloration reflects the degree of similarity among group members. In the case of VEGFA, both the coloration and block structure of ALI cases are most distinct with the quadratic function, and this is reflected in the most extreme p- value for the quadratic kernel test.