Haplotype Association Mapping of Acute Lung Injury in Mice Implicates Activin A Receptor, Type 1

Abstract

Rationale: Because acute lung injury is a sporadic disease produced by heterogeneous precipitating factors, previous genetic analyses are mainly limited to candidate gene case-control studies. Objectives: To develop a genome-wide strategy in which single nucleotide polymorphism associations are assessed for functional consequences to survival during acute lung injury in mice. Methods: To identify genes associated with acute lung injury, 40 inbred strains were exposed to acrolein and haplotype association mapping, microarray, and DNA-protein binding were assessed. Measurements and Main Results:The mean survival time varied among mouse strains with polar strains differing approximately 2.5-fold. Associations were identified on chromosomes 1, 2, 4, 11, and 12. Seven genes (Acvr1, Cacnb4, Ccdc148, Galnt13, Rfwd2, Rpap2, and Tgfbr3) had single nucleotide polymorphism (SNP) associations within the gene. Because SNP associations may encompass ''blocks'' of associated variants, functional assessment was performed in 91 genes within 6 1 Mbp of each SNP association. Using 10% or greater allelic frequency and 10% or greater phenotype explained as threshold criteria, 16 genes were assessed by microarray and reverse realtime polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-b signaling. Transcripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed between the strains with exposure and contained SNPs that could eliminate putative transcriptional factor recognition sites. Ccdc148, Fancl, and Tnn had sequence differences that could produce an amino acid substitution. Mycn and Mgat4a had a promoter SNP or 39untranslated region SNPs, respectively. Several genes were related and encoded receptors (ACVR1, TGFBR3), transcription factors (MYCN, possibly CCDC148), and ubiquitin-proteasome (RFWD2, FANCL, CACYBP) proteins that can modulate cell signaling. An Acvr1 SNP eliminated a putative ELK1 binding site and diminished DNA-protein binding. Conclusions: Assessment of genetic associations can be strengthened using a genetic/genomic approach. This approach identified several candidate genes, including Acvr1, associated with increased susceptibility to acute lung injury in mice.

Functional Genomic Assessment of Phosgene-Induced Acute Lung Injury in Mice

“…The G allele is the minor allele (15.3% allelic frequency) and could result in the gain of a putative DNA binding site for transcription factor 12. Expressed in the lung (64), transcription factor 12 can repress E-cadherin (cadherin 1, type 1, E-cadherin [epithelial]) (65) and may modulate SMAD signaling (66), which is often active during ALI by transforming growth factor (TGF)b1 (32,34).…”

“…The genetic and transcriptomic findings here may be limited to this compound and thus may not be relevant to other forms of ALI. We previously identified candidate genes associated with acrolein-induced (32,33) and chlorine-induced ALI (34), and these genes differed from those identified with phosgene. Until several types of chemically induced ALI have been evaluated, generalizations to other forms may not be warranted.…”

“…Until several types of chemically induced ALI have been evaluated, generalizations to other forms may not be warranted. Nonetheless, a major candidate identified previously with acrolein was Activin A receptor type 1 (Acvr1), which mediates TGF-b1 signaling (32). TGF-b1 signaling was also implicated with chlorine-induced ALI by the identification of Sema7a (34).…”

“…Genome-wide association analysis was performed using efficient mixed-models association corrected for confounding from population structure and genetic relatedness (43,44). Previously, we determined that one statistical association by chance will occur in a genome-wide scan when the threshold is decreased to 1.6 3 10 25 or 2log (P) ¼ 4.8 (25,(32)(33)(34). Therefore, we used a significance threshold of -log (P) .…”

“…Recently, nextgeneration whole genome sequencing information has been obtained on 17 mouse strains (29), which has strengthened the existing single nucleotide polymorphism (SNP) database for over 40 mouse strains (29)(30)(31). Using a genetically diverse panel of >40 inbred mouse strains, we previously conducted functional genomic analyses of acrolein-induced (32,33) and chlorine-induced (34) ALI. In this study, we sought to compare these findings with an additional chemical, phosgene, also known to produce ALI after accidental or intentional terrorist exposures.…”