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 ± 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 real-time polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-β 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 3'untranslated 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.

Figure 1.

Mouse strains vary in sensitivity to acrolein-induced acute lung injury. (A) Acute lung injury survival time of 40 mouse strains. Mice were exposed to 10 ppm acrolein for up to 24 hours and survival time recorded hourly. Values are mean ± SE (n = 6 to 16 mice/strain except MRL/MpJ, n = 3). (B) Haplotype association map for acrolein-induced acute lung injury in mice. The scatter (Manhattan) plot of corresponding −log(P) association probability for single nucleotide polymorphism (SNP) at indicated chromosomal location (ordinate). Transcripts with SNP associations of −log(P) > 4.0 were selected for further analysis. (C) Candidate genes on mouse chromosome 2 associated with acrolein-induced acute lung injury. The Manhattan plot of SNP associations indicates chromosomal location (ordinate) and corresponding –log(P) association probability. Genes with one or more significant SNP (−log[P] > 6.0) included Galnt13 (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalacto-aminyl transferase 13), Acvr1 (activin A receptor, type 1), and Ccdc148 (coiled-coil domain containing 148). Acvr1c = activin A receptor, type IC; Cytip = cytohesin 1 interacting protein; Galnt5 = UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetyl-galactosaminyl transferase 5; Gpd2 = glycerol phosphate dehydrogenase 2, mitochondrial; Kcnj3 = potassium inwardly-rectifying channel, subfamily J, member 3; Nr4a2 = nuclear receptor subfamily 4, group A, member 2.

Figure 2.

Histological assessment of lung tissue from (A) control SM/J mice, (B) control 129X1/SvJ mice, (C, E) acrolein-exposed SM/J mice, or (D, F) acrolein-exposed 129X1/SvJ mice. Consistent with acute lung injury, (C) perivascular enlargement (black arrow) and (E) leukocyte infiltration (red arrow) were more evident in the (C, E) sensitive SM/J strain than in the (D, F) resistant 129X1/SvJ strain. Mice were exposed to filtered air (control) or to acrolein (10 ppm, 17 h) and killed. Lung tissue was obtained, fixed in formaldehyde, and 5-μm sections prepared with hematoxylin and eosin stain. Bars indicate magnification.

Figure 3.

Characterization of acute lung injury by bronchoalveolar lavage. Mice were exposed to 10 ppm acrolein for 0 (filtered air control), 6, or 12 hours, killed, and bronchoalveolar lavage performed with (Ca²⁺, Mg²⁺ free) phosphate buffered saline. (A) Bronchoalveolar lavage protein increased sooner in the sensitive (SM/J) than in the resistant (129X1/SvJ) mouse strain. Lavage fluid was centrifuged and total protein in cell-free supernatants was measured using a bicinchoninic acid assay. (B) Bronchoalveolar lavage polymorphonuclear leukocytes increased in the sensitive (SM/J) but not in the resistant (129X1/SvJ) mouse strain. After centrifugation, cell pellet was suspended and an aliquot (200 μl) were cytocentrifuged and the cells were stained with Hemacolor for differential cell analysis according to standard cytological procedures. (C) Bronchoalveolar lavage nitrite concentration increased sooner in the sensitive (SM/J) than in the resistant (129X1/SvJ) mouse strain. Supernatant was analyzed using a fluorometric method in which nitrite reacted with 2,3-diaminonaphthalene. *Significantly different from strain-matched control as determined by analysis of variance with an all pairwise multiple comparison procedure (Holm-Sidak method). ^†Significantly different between the sensitive SM/J and resistant 129X1/SvJ mouse strain as determined by analysis of variance with an all pairwise multiple comparison procedure (Holm-Sidak method).

Figure 4.

Pathways enriched in transcripts in sensitive (SM/J) and resistant (129X1/SvJ) mouse lung during acrolein exposure. Mice were exposed to 10 ppm acrolein for 0 (filtered air control), 6, or 12 hours, killed, and lung tissue frozen in liquid nitrogen. Lung transcript levels were then quantified by microarray and compared with filtered air control. Pathway enrichment was determined using significant values (> twofold and P < 0.01 by analysis of variance) analyzed by Ingenuity Pathways Analysis. The top three enriched pathways/lists for “Canonical pathway,” “Biological function,” or “Toxicology list” categories were selected based on the combined 6- and 12-hour −log(P). The strain- and time-specific −log(P) value is presented in parentheses. Left: Increased transcripts were enriched in (top) transforming growth factor-β (TGFB) signaling, (middle) cell death, and (bottom) nuclear factor, erythroid derived 2, like 2 (NFE2L2)-mediated oxidative stress. Increased transcripts in TGFB signaling (e.g., SM/J –log(P) = 5.8 vs. 129X1/SvJ –log(P) = 1.2 at 12 h) and cell death pathway (e.g., SM/J –log(P) = 24.6 vs. 129X1/SvJ –log(P) = 15.4 at 12 h) were increased more in SM/J as compared with 129X1/SvJ mouse strains. In contrast, the response of SM/J and 129X1/SvJ were similar in the NFE2L2-mediated oxidative stress response (e.g., SM/J –log(P) = 5.8 vs. 129X1/SvJ –log(P) = 6.0 at 12 h). Right: Decreased transcripts were enriched in (top) glucocorticoid receptor signaling, (middle) lipid metabolism, and (bottom) retinoic acid receptor-α (RAR) activation. The responses in SM/J and 129X1/SvJ mouse strains were similar (except DnaJ [Hsp40] homolog, subfamily A, member 1 [DNAJA1] and stress-induced phosphoprotein 1 [STIP1]) in these pathways. Bars are mean ± SE (n = 5 mice/strain/time). Additional abbreviations: see Methods section in online supplement.

Figure 5.

Assessment of basal transcript levels of 16 candidate genes for acrolein-induced acute lung injury in mice. Mice were exposed to filtered air (control) and lung mRNA isolated. Basal transcript levels of SM/J (sensitive) mouse strain were compared with those of 129X1/SvJ (resistant) mouse strain as determined by quantitative real-time polymerase chain reaction. Values are mean ± SE of the transcript level of SM/J (n = 8) as compared with the 129X1/SvJ (n = 8). *Significantly different between the sensitive SM/J and resistant 129X1/SvJ mouse strain as determined by analysis of variance with an all pairwise multiple comparison procedure (Holm-Sidak method). ACVR1 = activin A receptor, type 1; ARHGAP15 = Rho GTPase activating protein 15; CACNB4 = calcium channel, voltage-dependent, β 4 subunit; CACYBP = calcyclin binding protein; CCDC148 = coiled-coil domain containing 148; FANCL = Fanconi anemia, complementation group L; GALNT13 = UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyl transferase 13; MGAT4A = mannoside acetylglucosaminyltransferase 4, isoenzyme A; MYCN = v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian); RBMS1 = RNA binding motif, single stranded interacting protein 1; RFWD2 = Ring finger and WD repeat domain 2; RPAP2 = RNA polymerase II associated protein 2; STAM2 = signal transducing adaptor molecule (SH3 domain and ITAM motif) 2; TANC1 = tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 1; TGFBR3 = transforming growth factor, β receptor III; TNN = tenascin N.

Figure 6.

Transcript levels of six candidate genes that differed between the SM/J and 129X1/SvJ mouse strains after acrolein exposure. Mice were exposed to filtered air (control, 0 h) or to acrolein (10 ppm) for 6 or 12 hours, lung mRNA isolated, and transcript expression levels determined by quantitative real-time polymerase chain reaction. Values are mean ± SE of the transcript level of SM/J (n = 8) as compared with the 129X1/SvJ (n = 7–8). *Significantly different from strain-matched control as determined by analysis of variance with an all pairwise multiple comparison procedure (Holm-Sidak method). ^†Significantly different between the sensitive SM/J and resistant 129X1/SvJ mouse strain as determined by analysis of variance with an all pairwise multiple comparison procedure (Holm-Sidak method). ACVR1 = activin A receptor, type 1; ARHGAP15 = Rho GTPase activating protein 15; CACNB4 = calcium channel, voltage-dependent, β 4 subunit; CACYBP = calcyclin binding protein; RFWD2 = Ring finger and WD repeat domain 2; TGFBR3 = transforming growth factor-β receptor III.

Figure 7.

Single nucleotide polymorphism (rs6406107) in the 5′ untranslated region of Acvr1 diminishes nuclear protein binding capacity. (A) Oligonucleotide probe containing the G-allele, in contrast to the A-allele, formed a distinct fast-migrating complex (lower band). Electrophoretic mobility shift assay (EMSA) of nuclear protein extract prepared from mouse lung epithelial cells (MLE-15) and biotinylated oligonucleotide probes containing the A- or G-allele. (B) Compared with the A-variant, the G-variant is a more effective competitor of protein-biotinylated DNA complexes formation. Competitive EMSA was performed with excess double-strained oligonucleotides that contained either the A- or G-variant. The biotinylated labeled rs6406107 A-variant oligonucleotide probe is readily competed by nonbiotinylated rs6406107 A- or G-variant, whereas the biotinylated labeled rs6406107 G-variant oligonucleotide probe is more avidly bound, in particular the fast migrating complex (right, lane 2).