Haplotype Association Mapping of Acute Lung Injury
in Mice Implicates Activin A Receptor, Type 1
George D. Leikauf1, Vincent J. Concel1, Pengyuan Liu2, Kiflai Bein1, Annerose Berndt3, Koustav Ganguly1,
An Soo Jang1,4, Kelly A. Brant1, Maggie Dietsch5, Hannah Pope-Varsalona1, Richard A. Dopico, Jr.1,
Y. P. Peter Di1, Qian Li5, Louis J. Vuga6, Mario Medvedovic5, Naftali Kaminski6, Ming You2, and Daniel R. Prows7,8
1Department of Environmental and Occupational Health, Graduate School of Public Health,3Department of Medicine,6Simmons Center for
Interstitial Lung Disease, University of Pittsburgh, Pittsburgh, Pennsylvania;2Wisconsin Cancer Center, Medical College of Wisconsin, Milwaukee,
Wisconsin;4Department of Internal Medicine, Soon Chun Hyang University, Bucheon, Korea;5Department of Environmental Health, and
7Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio; and8Division of Human Genetics, Cincinnati Children’s Hospital, Cincinnati, Ohio
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.
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
allelicfrequencyand 10% orgreaterphenotypeexplained asthresh-
old criteria, 16 genes were assessed by microarray and reverse real-
pathways including transforming growth factor-b signaling. Tran-
scripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed be-
tween 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
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
Conclusions: Assessment of genetic associations can be strengthened
using a genetic/genomic approach. This approach identified several
candidate genes, including Acvr1, associated with increased suscepti-
bility to acute lung injury in mice.
Keywords: acute respiratory distress syndrome; smoke inhalation; car-
boxyl stress; transforming growth factor-&beta signaling; ubiquitination
Acute lung injury is marked by pulmonary edema resulting from
increased epithelial and endothelial permeability, decreased clear-
ance of edema fluid, and disruption of surfactant-associated pro-
tein function (1–3). This condition can be induced directly (e.g.,
through smoke inhalation) or indirectly (e.g., through sepsis) (1).
In smoke inhalation, the severity of acute lung injury (2) and car-
diovascular dysfunction (4) are critical determinants of morbidity
and mortality (5–7). Acrolein, a potent irritant (8–10), is the major
chemical in smoke responsible for pulmonary edema (11, 12).
A major challenge to critical care is to reliably predict and
enhance survival in acute lung injury (7). Individual suscepti-
bility varies greatly (i.e., patients presenting with the same
severity score can have markedly different clinical outcomes)
(13). For this reason, studies have begun to investigate the role
of genetics in determining survival during acute lung injury (14–
17). However, because acute lung injury is a sporadic disease
produced by heterogeneous precipitating factors, previous
genetic analyses are mainly limited to case-control studies that
evaluate candidate genes associations.
Constant acquisition of genome-wide information on numer-
ous species, including more than 40 mouse strains (18–21),
makes the mouse a useful model to facilitate rapid evaluation of
the genetic basis of human physiology and pathophysiology (22,
23). Complementing conventional single gene mapping ap-
proaches, genome-wide mapping has mainly used quantitative
trait locus (QTL) analysis in mice, which has been a valued tool
to identify candidate genes responsible for complex traits un-
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
A major challenge to critical care is to reliably predict
survival of patients with acute lung injury. Individual
susceptibility varies greatly (i.e., patients presenting with
the same severity score can have markedly different clinical
outcomes). For this reason, studies have begun to investigate
the role of genetics in determining survival during acute lung
injury. However, because acute lung injury is a sporadic
disease produced by heterogeneous precipitating factors,
previous genetic analyses are mainly limited to case-control
studies that evaluate candidate genes associations.
What This Study Adds to the Field
This study presents a strategy in which single nucleotide poly-
morphism associations are assessed for functional conse-
quences to survival during acute lung injury in mice. This
approach identified several candidate genes, and Acvr1 may be
particularly worthy of future investigations in acute lung injury.
(Received in original form June 15, 2010; accepted in final form February 4, 2011)
Supported by National Institutes of Health grants ES015675, HL077763, and
HL085655 (G.D.L); HL091938 (Y.P.P.D.), HG003749 and LM009662 (M.M.);
HL084932 and HL095397 (N.K.); AT003203, AT005522, CA113793, and
CA134433 (M.Y.); and HL075562 (D.R.P.).
Correspondence and requests for reprints should be addressed to George D.
Leikauf, Ph.D., Department of Environmental and Occupational Health, Graduate
School of Public Health, University of Pittsburgh, 100 Technology Drive, Suite
350, Pittsburgh, PA 15219-3130. E-mail: email@example.com
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.201006-0912OC on February 25, 2011
Internet address: www.atsjournals.org
Vol 183. pp 1499–1509, 2011
derlying human disease (24–26). However, a major obstacle of
identifying genes is the difficulty of resolving large QTL regions
(10–20 cM) into sufficiently small intervals to make positional
cloning practical (27). In this study, haplotype association map-
ping is used to obtain a dense single-nucleotide polymorphism
(SNP) map, which offers finer genetic resolution in the identifi-
cation of genetic determinants of acute lung injury.
This study was performed in accordance with the Institutional Animal
Care and Use Committee of the University of Pittsburgh (Pittsburgh,
PA) and mice were housed under specific pathogen-free conditions.
Forty inbred strains (n 5 507 mice, 6–8 wk old, female; The Jackson
Laboratory, Bar Harbor, ME) were used in the study. Mice were ex-
posed to filtered air (control) or acrolein (10 ppm, 24 h) as generated
and monitored as described previously (8), and survival time was
recorded. Haplotype association analysis was performed using thresh-
old –log(P) value of 6.0 for significant and 4.0 for suggestive linkage as
described previously (23). To examine acrolein-induced changes in
lung histology, bronchoalveolar lavage, and lung transcripts, 129X1/SvJ
and SM/J mice (two strains that represented the opposite ends of the
phenotypic spectrum of the analyzed strains) were exposed to filtered
air (0 h, control) or acrolein (10 ppm for 6, 12, or 17 h). Microarray
analysis and quantitative real time-polymerase chain reaction (qRT-
PCR) were used to determine transcript levels of genes previously asso-
ciated with acute lung injury and to contrast genes identified. Three 59
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 6 SE (n 5 6 to 16 mice/strain
except MRL/MpJ, n 5 3). (B) Haplotype association map
for acrolein-induced acute lung injury in mice. The scatter
(Manhattan) plot of corresponding 2log(P) association
probability for single nucleotide polymorphism (SNP) at
indicated chromosomal location (ordinate). Transcripts
with SNP associations of 2log(P) . 4.0 were selected for
further analysis. (C) Candidate genes on mouse chromo-
some 2 associated with acrolein-induced acute lung injury.
The Manhattan plot of SNP associations indicates chro-
mosomal location (ordinate) and corresponding –log(P)
association probability. Genes with one or more significant
SNP (2log[P] . 6.0) included Galnt13 (UDP-N-acetyl-a-D-
galactosamine:polypeptide N-acetylgalacto-aminyl trans-
ferase 13), Acvr1 (activin A receptor, type 1), and Ccdc148
(coiled-coil domain containing 148). Acvr1c 5 activin A
receptor, type IC; Cytip 5 cytohesin 1 interacting protein;
Galnt5 5 UDP-N-acetyl-a-D-galactosamine:polypeptide
N-acetyl-galactosaminyl transferase 5; Gpd2 5 glycerol
phosphate dehydrogenase 2, mitochondrial; Kcnj3 5
potassium inwardly-rectifying channel, subfamily J, mem-
ber 3; Nr4a2 5 nuclear receptor subfamily 4, group A,
Mouse strains vary in sensitivity to acrolein-
1500AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1832011
untranslated region (UTR) SNPs in Acvr1 were genotyped in 28 strains
and the consequences of the rs6406107 variant on DNA–protein binding
was assessed by electrophoretic mobility shift assay. Additional details of
the methods are presented in the online supplemental.
Survival Time of 40 Mouse Strains during Acute Lung Injury
Mice from 40 mouse strains were exposed to acrolein and sur-
vival time recorded. The mean survival time varied among
mouse strains (Figure 1A). The most polar strains varied ap-
proximately 2.5-fold from 16.5 6 0.9 hours (MOLF/EiJ) to
41.1 6 4.5 hours (129X1/SvJ). Using the data from all the mice,
a haplotype association map was obtained for acrolein-induced
acute lung injury (Figure 1B). Significant [2log(P) > 6.0] as-
sociations were identified on chromosome 2 with suggestive
[2log(P) > 4.2] associations identified on chromosomes 1, 2, 4,
11, and 12. Five genes (Rfwd2, Cacnb4, Galnt13, Acvr1, and
Rpap2) had three to four significant or suggestive SNP associ-
ations within the gene boundary, whereas two genes (Ccdc148
and Tgfbr3) had one significant or suggestive SNP association
within the gene boundary (see Table E1 in the online supple-
ment). Evaluation of SNP associations determined the allelic
frequency ranged from 0.20 to 0.54 (or 73 and 197 mice, re-
spectively) among the 40 strains tested for survival (n 5 365
mice). Because a sufficient number of mice carried the ‘‘minor’’
allele (> 10%), the percentage of phenotype explained by each
SNP association was determined using the difference between
the mean survival time of mice carrying either allele and di-
viding it by the total difference between the two polar strains
(24.6 h) (Figure E1). The amount of phenotype explained by the
SNP associations was substantial (range: 17–34%) (Table E1).
Candidate Genes on Chromosome 2 Associated
with Acrolein-induced Acute Lung Injury
A number of SNPs with association probabilities 2log(P) of 5.0
to 7.0 were identified in a small region on chromosome 2 (54–59
Mbp). Genes in this region with one or more significant SNP
associations included Galnt13, Acvr1, and Ccdc148 (Figure 1C).
Because any SNP association may encompass blocks of associ-
ated variants that reside near the identified SNP (28), functional
assessment was performed on SNPs within 91 genes that mapped
within 1 Mbp of a significant/suggestive SNP association. Using
10% or greater allelic frequency and 10% or greater phenotype
explained as threshold criteria, 16 candidate genes qualified for
Histological, Bronchoalveolar Lavage, and Transcriptional
Assessment of Acrolein-induced Lung Tissue in SM/J
and 129X1/SvJ Mouse Strains
To further assess susceptibility, a sensitive and a resistant mouse
strain were contrasted by detailed characterization. For the
sensitive strain, we selected SM/J and avoided wild-derived
(e.g., MOLF/EiJ, SPRET/EiJ, or PERA/EiJ) or repository (e.g.,
RIIIS/J) strains. The mean survival time for the SM/J strain
(17.2 6 0.46 h) was not significantly different from that of
MOLF/EiJ. The 129X1/SvJ was selected as representative of
a resistant strain. Histological analyses of these strains were
consistent with acute lung injury. Perivascular enlargement and
leukocyte infiltration were more evident in the sensitive SM/J
strain than in the resistant 129X1/SvJ strain (Figure 2). Simi-
larly, bronchoalveolar lavage protein concentration (Figure
3A), polymorphonuclear leukocyte percentage (Figure 3B),
and nitrite concentration (Figure 3C) increased sooner in the
SM/J than in 129X1/SvJ mice.
Lung transcript levels were also compared between SM/J
and 129X1/SvJ strains using microarray analysis (Table E2).
Previously identified transcripts that are altered in mouse lung
during acute lung injury include IL-6 (29) and metallothionein 2
(MT2) (30). Microarray results indicated that IL6 transcripts at
6 or 12 hours increased more in SM/J than in 129X1/SvJ lung
(Figure E2, top). These differences were confirmed by qRT-
PCR (although microarray increases were less than qRT-PCR
increases). In contrast, MT2 transcripts increased in both strains
when compared with strain-matched controls but were not
different between the strains (Figure E2, bottom).
Further evaluation of microarray transcripts that increased
with exposure (fold log 2 > 1.0; P , 0.01) revealed enriched
pathways including transforming growth factor-b (TGFB) sig-
naling, cell death, and nuclear factor, erythroid derived 2, like 2
(NFE2L2)-mediated oxidative stress response for transcripts
that increased with exposure (Figure 4; Table 1). Transcripts
in TGFB signaling (e.g., SM/J 2log(P) 5 5.8 vs. 129X1/SvJ
2log(P) 5 1.2 at 12 h) or cell death pathway (e.g., SM/J 2log(P) 5
24.6 vs. 129X1/SvJ –log(P) 5 15.4 at 12 h) were increased more
in SM/J as compared with 129X1/SvJ mice. 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 5.8 vs.
129X1/SvJ –log(P) 5 6.0 at 12 h). The latter is noteworthy in
that NFE2L2 activation leads to accumulation of transcripts
encoding enzymes that may be viewed as protective (e.g., heme
oxygenase [decycling] 1 [HMOX1]) during acute lung injury
(31). One transcript in the TGFB signaling pathway, secreted
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-mm sections pre-
pared with hematoxylin and eosin stain. Bars indicate magnification.
Leikauf, Concel, Liu, et al.: Genetics of Acute Lung Injury1501
phosphoprotein 1 (SPP1), was also evaluated using qRT-PCR.
At 6 or 12 hours, SPP1 transcript increased in SM/J mice, but
was not significantly different from control in 129X1/SvJ mice
(Figure E3). The increase at 12 hours was significantly greater in
the SM/J lung as compared with the 129X1/SvJ lung.
For microarray transcripts that decreased with exposure,
enriched pathways included glucocorticoid receptor signaling,
lipid metabolism, and retinoic acid receptor activation (Figure
4). In general, pathways with decreased transcripts were similar
between these strains as determined by the enrichment proba-
bility 2log(P). Individual transcripts among these pathways
were decreased more in the SM/J than in 129X1/SvJ mice,
Additional pathways that were enriched in SM/J but were
not in 129X1/SvJ mice included (1) increased transcripts: IL-10
signaling, mTOR signaling, and immune cell trafficking; and (2)
decreased transcripts: lysine biosynthesis (Table 1). Pathways
that were enriched in 129X1/SvJ but not in SM/J mice included
(1) increased transcripts: phospholipid degradation, sphingo-
sine-1-phosphate signaling, protein ubiquitination pathway, cell
movement, and antiapoptosis; and (2) decreased transcripts:
ephrin receptor signaling.
Assessment of Basal Transcript Levels of 16 Candidate Genes
for Acrolein-induced Acute Lung Injury in Mice
Having found that SM/J and 129X1/J mice differ in the time-
course of acrolein-induced lung injury, the identified 16 candi-
date genes were then evaluated by qRT-PCR. Initially, basal
levels were compared and transcript levels of 11 of the 16 genes
were significantly different between strains (Figure 5).
Next, the time course of transcripts of the 16 candidate genes
was measured during exposure and compared with the strain-
matched control. This analysis identified six transcripts (ACVR1,
ARHGAP15, CACNB4, CACYBP, RFWD2, and TGFBR3)
that were different between the strains with exposure (Figure
6). For each of these transcripts, the corresponding gene was
interrogated to determine whether it contained SNPs that could
alter putative transcriptional factor recognition sites (i.e., ex-
pression SNP or eSNP). Examining the sequence variability
within 22,000 bp in the 59UTR through intron 1, five genes,
Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3, were identified
to have eSNPs that could eliminate ELK1, GAL4, HES-1, YY1,
and NF-1 binding motifs as determined by Transcription Element
Search Software (32), respectively (Table 2). The survival time
associated with either allele was then determined for the mouse
population. Mice possessing these eSNPs exhibited mean survival
time of 17–30% of the difference between the mean survival times
of the most polar strains. The sixth gene, Cacnb4, lacked an eSNP
associated with a difference in phenotype in the 59UTR but con-
tained an SNP that would produce a variant splicing transcript.
In addition, four transcripts (CCDC148, FANCL, MYCN,
and TNN) were altered by exposure but were not different
between the strains (Figure E4). The corresponding genes were
then interrogated to determine whether they contained a coding
nonsynonymous SNP (i.e., cnSNP) that could produce amino
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 (Ca21, Mg21free) 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 bicincho-
ninic acid assay. (B) Bronchoalveolar lavage polymorphonuclear leuko-
cytes 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 ml) were cytocentrifuged and the cells were stained
with Hemacolor for differential cell analysis according to standard
cytological procedures. (C) Bronchoalveolar lavage nitrite concentra-
tion increased sooner in the sensitive (SM/J) than in the resistant
(129X1/SvJ) mouse strain. Supernatant was analyzed using a fluoro-
metric 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-
1502AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1832011
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 2log(P). The strain- and time-specific 2log(P) value is presented in parentheses. Left: Increased transcripts were
enriched in (top) transforming growth factor-b (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 5.8 vs. 129X1/SvJ –log(P) 5 1.2 at 12 h) and cell death pathway (e.g., SM/J
–log(P) 5 24.6 vs. 129X1/SvJ –log(P) 5 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 5.8 vs. 129X1/SvJ –log(P) 5 6.0 at 12 h). Right:
Decreased transcripts were enriched in (top) glucocorticoid receptor signaling, (middle) lipid metabolism, and (bottom) retinoic acid receptor-a (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 mean6 SE (n 5 5 mice/strain/time). Additional abbreviations: see Methods section in online supplement.
Leikauf, Concel, Liu, et al.: Genetics of Acute Lung Injury1503
acid changes. Two genes, Ccdc148 and Fancl, had Gln81Arg
and Asp1Ala amino acid substitution, respectively (Table 2).
Although lacking a cnSNP, Mycn had an eSNP that could eliminate
a MTF-1 binding motif. In Tnn, three SNPs (rs4877414, rs46709434,
and rs49224805) could produce Ala310Thr, Lys333Gln, and
Arg562Cys amino acid substitutions, respectively. Mice having
either allele in the identified SNPs exhibited phenotypic differ-
ences in survival of 11 to 28%.
The predicted amino acid substitutions (missense mutations)
were examined using Prosite (33, 34) to determine whether they
are situated in functional domains of the encoded protein. In
CCDC148, the rs27956803 Gln81Arg substitution resulted in
a gain of a predicted protein kinase C phosphorylation site at
amino acid 79 to 81. The Asp1Ala substitution in FANCL was
not in a predicted functional domain. In TNN, three amino acid
substitutions are located in fibronectin type III domains and the
Ala310Thr substitution resulted in a loss of a predicted protein
kinase C phosphorylation site.
Last, MGAT4A transcripts did not change significantly with
exposure in either strain of mouse tested. As noted above, basal
transcript levels were different between the strains. The corre-
sponding sequence was therefore interrogated for eSNP and
cnSNP, and none were observed. The 39UTR was then exam-
ined and an SNP was observed in exon 15/39-UTR, which is in
a region that may alter message stability.
Together, the difference in transcript levels combined with the
in silico sequence assessment yielded 11 candidates (Mgat4a,
Rfwd2, Tnn, Cacybp, Arhgap15, Cacnb4, Acvr1, Ccdc148, Tgfbr3,
Fancl, and Mycn) with probable functional associations to di-
minished survival resulting from acrolein-induced acute lung in-
jury (Table 2). Candidate genes with functional SNP associations
were located in eight chromosomal regions (Mbp): 1 (37), 1 (161),
2 (43), 2 (52), 2 (58), 5 (107), 11 (26), and 12 (12) and indepen-
dently could explain 11 to 30% of the phenotype.
Acvr1 Promoter Analysis
Three SNPs (rs33408603, rs6406068, and rs6406107) were iden-
tified in the 59UTR region of Acvr1. However, few strains had
previously been genotyped (129X1/SvJ, C57BL/6J, and A/J for
rs33408603, and CZECHII/EiJ and C57BL/6J for rs6406068 or
rs6406107). To obtain additional genotype information on these
SNPs, 28 strains were genotyped by PCR amplification–DNA
sequencing (Table E3). One SNP, rs33408603 (A/G), was ob-
served only in the 129X1/SvJ strain (A-allele). The remaining
SNPs exhibited an identical haplotype in all strains typed (i.e.,
strains with the rs6406068 C-allele had the rs6406107 A-allele).
The allele frequency of this SNP haplotype was 12% and could
explain 19% of the phenotype. Analysis of the possible conse-
quence of these SNPs indicated that the rs6406107 A-allele
would result in the loss of a putative ELK1, member of ETS
oncogene family (ELK1) transcription factor binding site. In
vitro, an Acvr1 promoter region–derived oligonucleotide con-
taining the rs6406107 G-variant more effectively competed the
nuclear protein-binding capacity of a labeled probe than that
containing the A-variant (Figure 7).
Past studies using candidate gene approaches have been prom-
ising and have provided valuable insights into the genetic
architecture of the complex trait of acute lung injury; however,
these studies also have had recognized limitations (28, 35–37).
For several reasons it has been difficult to assemble a large
enough cohort of clinically identical individuals to perform a
genome-wide association study in humans. Adding to this bar-
rier are the difficulties in establishing functional significance of
genetic associations (38). Thus, using genome-wide association
approaches to identification of candidate genes and related
TABLE 1. ENRICHED PATHWAYS OF LUNG TRANSCRIPTS IN SENSITIVE (SM/J) AND RESISTANT (129X1/SVJ) MOUSE STRAINS EXPOSE
Immune cell trafficking
NFE2L2-mediated oxidative stress response
Glucocorticoid receptor signaling
Cardiovascular system development and function
Respiratory system development and function
Cytochrome P450 panel - substrate is an eicosanoid
6 h12 h Increased
Protein ubiquitination pathway
NFE2L2-mediated oxidative stress response
Ephrin receptor signaling
Glucocorticoid receptor signaling
Cardiovascular system development and function
Respiratory system development and function
Cytochrome P450 panel - substrate is an eicosanoid
6 h12 h
Definition of abbreviations: NFE2L2 5 nuclear factor, erythroid derived 2, like 2; RAR 5 retinoic acid receptor.
Mice (n 5 5 mice/strain/time) were exposed to 10 ppm acrolein, killed, lung removed, and RNA isolated. Transcript levels were then quantified by microarray and
compared to filtered air control. Pathway enrichment was determined using significant values (. twofold and P , 0.01 by analysis of variance) with Ingenuity Pathways
Analysis (Ingenuity Systems, www.ingenuity.com). The top three enriched pathways for ‘‘Canonical pathway,’’ ‘‘Biological function,’’ and ‘‘Toxicology list’’ categories
were selected for increased and decreased expression changes, based on the 2log(P) for the combined 6- and 12-h data.
1504 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1832011
pathways (39) in mice could be a valued additional approach
Previously, considerable interstrain differences have been
reported in acute lung injury susceptibility (24, 25). In this study
with acrolein, we observed an approximately 2.5-fold difference
in survival time between the most sensitive and resistant strains,
and thus this trait was amenable to mapping. In past decades,
QTL mapping has identified chromosomal regions containing
genes affecting cancer, diabetes, hypertension, and other disease-
related phenotypes. However, a major obstacle of identifying
QTL genes is the difficulty of resolving large chromosomal
regions (10–20 cM) into sufficiently small intervals to make po-
sitional cloning practical. With advances in genome sequencing,
dense SNP maps have proven successful in the refinement of
previous QTL regions and the identification of new genetic
determinants of complex traits (21).
One concern about any SNP association study is that variants
identified in genome-wide association study may confer a rela-
tively small increment of risk (explaining 1–3% to the popu-
lation variance) (28, 35–38). Here, we present a strategy to
overcome this limitation in which SNPs within 1 Mbp of an
association are assessed for functional consequences to survival
in the mice tested. A threshold of 10% or greater survival time
between the most sensitive and resistant strains was set for
further assessment of candidates. Type I error was diminished
by comparing the phenotype (i.e., individual survival time) of
every member of the population with the corresponding strain
genotype. This approach also limits the associated SNPs to
those with feasible functional consequence to expression (eSNP)
or amino acid sequence (cnSNP).
Of the candidate genes identified, only Mycn has been asso-
ciated with respiratory failure. Mice with a Mycn mutation (that
reduces expression through alternative splicing) die at birth due
to respiratory failure (40). However, this effect is probably due
to defective lung development (41) and abnormal branching
morphogenesis (42), because complete ablation of the gene is
embryonic lethal. The other candidate genes identified in this
study have not previously been associated with acute lung injury.
Nonetheless, these genes are present in the lung and the cor-
responding proteins have functions related to the cell stress
signaling in lung injury.
As with any study, this investigation has several limitations.
Although acrolein-induced acute lung injury has relevance to
smoke inhalation, numerous other agents (either chemical or
infectious) can elicit acute lung injury (43). Until these other
forms or phenotypes (e.g., lavage protein) of acute lung injury
are evaluated, generalization to other forms of this condition is
not warranted. Supportive evidence by another genetic ap-
proach (e.g., traditional back-cross or F2 cohort) that likewise
identified the candidate genes identified with this haplotype
association mapping analysis would strengthen the associations
found in this study. In addition, although this approach may
improve the assessment of the results obtained from a haplotype
mapping analysis, functional assessment of each candidate gene
will require further studies (e.g, gene-targeted or transgenic mice).
Last, animal models are limited in that species differences in lung
structure and function can diminish applicability to humans. De-
spite these limitations, the identified genes and related pathways
may help to direct future human genetic studies that evaluate such
pathways using selected tagSNPs.
One of the leading candidate genes was Acvr1. In mice,
Acvr1 gene targeting produces a morphological gastrulation
defect, which is embryonic lethal (44–46). In humans, mutations
in the glycine-serine–rich (GS) activation domain or sites that
interact with the GS domain of ACVR1 have been associated
with fibrodysplasia ossificans progressiva, which is characterized
by progressive heterotopic ossification that can lead to re-
spiratory difficulties (47, 48). The ACVR1 GS domain is in-
volved in phosphorylation and human mutations lead to a gain
of function (i.e., augmented signaling) (49). In mice, postnatal
expression of mutant ACVR1 can lead to ectopic ossification,
but only when combined with infection. Corticosteroid inhibited
ossification in mice, suggesting that mutant ACVR1 and in-
flammation are both required for ectopic ossification (50).
Although named activin A receptor, type 1 (a.k.a. activin
receptor-like kinase 2, Alk2), ACVR1 does not bind activins
(a.k.a. inhibins), but binds BMP2, 4, 6, and 7, through a hetero-
meric complex with BMPR2 (51–54) (Figure E5, Table E4).
Critical to respiratory organogenesis and development (55, 56),
BMPs elicit various effects in adult tissues through type I and
II receptors, which in turn phosphorylate receptor-regulated
R-SMAD protein (primarily SMAD1, 5, and 8) (57, 58). On
activation, R-SMAD proteins associate with the common medi-
ator SMAD4 and translocate to the nucleus, where they act as
transcription factors to regulate expression of target genes, in-
cluding the inhibitory SMADs (I-SMAD), SMAD6, and SMAD7
(59). SMAD6 and SMAD7 inhibit/modulate TGFB/BMP signal-
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 poly-
merase chain reaction. Values are mean 6 SE of the transcript level of
SM/J (n 5 8) as compared with the 129X1/SvJ (n 5 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 5 activin A receptor,
type 1; ARHGAP15 5 Rho GTPase activating protein 15; CACNB4 5
calcium channel, voltage-dependent, b 4 subunit; CACYBP 5 calcyclin
binding protein; CCDC148 5 coiled-coil domain containing 148;
FANCL 5 Fanconi anemia, complementation group L; GALNT13 5
transferase 13; MGAT4A 5 mannoside acetylglucosaminyltransferase
4, isoenzyme A; MYCN 5 v-myc myelocytomatosis viral related
oncogene, neuroblastoma derived (avian); RBMS1 5 RNA binding
motif, single stranded interacting protein 1; RFWD2 5 Ring finger and
WD repeat domain 2; RPAP2 5 RNA polymerase II associated protein 2;
STAM2 5 signal transducing adaptor molecule (SH3 domain and ITAM
motif) 2; TANC1 5 tetratricopeptide repeat, ankyrin repeat and coiled-
coil containing 1; TGFBR3 5 transforming growth factor, b receptor III;
TNN 5 tenascin N.
Leikauf, Concel, Liu, et al.: Genetics of Acute Lung Injury1505
ing by interfering with the activation of other SMADS (59–61),
thereby providing negative feedback control (62).
In this study we found that lung ACVR1 transcripts de-
creased more in the sensitive SM/J than the resistant 129X1/SvJ
mice. In vitro, an Acvr1 promoter region SNP rs6406107 di-
minished nuclear protein-binding capacity of a labeled probe
and was associated with decreased survival in mice. This SNP
could lead to diminished ACVR1 transcript levels because the
A-allele would eliminate a putative ELK1 binding site. ELK1
can be phosphorylated by a TNF-initiated, JUN-mediated mech-
anism in pulmonary epithelial cells (63). A possible consequence
of decreased ACVR1 transcripts would be augmented TGFB/
BMP signaling. This is consistent with increases in transcripts
encoded by TGFB target genes noted in the microarray. Pre-
viously, TGFB/BMP signaling has been associated with adverse
effects during acute lung injury (64–66). Active TGFB1 is in-
creased in edema fluid obtained from patients with acute lung
injury (67–70), and TGFB1 decreases pulmonary endothelial
(71) or epithelial integrity (72) and diminishes epithelial fluid
transport (73, 74). The determination whether ACVR1 may
serve to limit TGFB/BMP signaling during lung injury is worthy
of future investigation.
Compared with 129X1/SvJ mice, several stress-response tran-
scripts (e.g., CACYBP, DNAJA1, FKBP4, HSPA1A, NFAT5,
and STIP1) decreased more in the SM/J lung. Inasmuch as these
transcripts encoded chaperon proteins, a decrease could de-
stabilize existing proteins (i.e., aggregation and unfolding of
newly translated proteins) and diminish the effectiveness of
ubiquitin-proteasome pathway (75, 76). Ubiquitin-proteasome
proteins may interact with other candidates identified (77). For
candidate genes that differed be-
tween the SM/J and 129X1/SvJ
mouse strains after acrolein expo-
sure. Mice were exposed to fil-
tered air (control, 0 h) or to
acrolein (10 ppm) for 6 or 12
hours, lung mRNA isolated, and
transcript expression levels deter-
mined by quantitative real-time
polymerase chain reaction. Values
are mean 6 SE of the transcript
level of SM/J (n 5 8) as compared
with the 129X1/SvJ (n 5 7–8).
*Significantly different from strain-
matched control as determined
by analysis of variance with an
all pairwise multiple comparison
procedure (Holm-Sidak method).
Transcript levels of six
†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 pro-
cedure (Holm-Sidak method).
ACVR1 5 activin A receptor, type
1; ARHGAP15 5 Rho GTPase acti-
vating protein 15; CACNB4 5
calcium channel, voltage-depen-
dent, b 4 subunit; CACYBP 5 cal-
cyclin binding protein; RFWD2 5
Ring finger and WD repeat domain
2; TGFBR3 5 transforming growth
factor-b receptor III.
1506 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1832011
example, ACVR1 interacts with FANCL (78) and modulates
SMURF2 ubiquitination of various proteins, including SMADs.
Two candidate genes, Rfwd2 and Cacybp, encode proteins with
ubiquitin-protein ligase activity. Besides limiting TP53 (a.k.a.
p53) (79), RFWD2 suppresses JUN (80) and FOXO1 (81) and
other transcription factor accumulation and thereby limits cell
stress (82). For example, JUN diminishes surfactant-associated
protein B synthesis and thereby increases mortality during acute
lung injury (3, 83), and FOXO1 is critical to maintaining claudin
5 in acrolein-induced acute lung injury (84).
In summary, haplotype association mapping, microarray/
qRT-PCR analyses, and in silico SNP analysis identified 11
candidate genes (Acvr1, Arhgap15, Cacnb4, Cacybp, Ccdc148,
Fancl, Mycn, Mgat4a, Rfwd2, Tgfbr3, and Tnn) associated with
acrolein-induced acute lung injury in mice. Several genes were
related and encoded receptors (ACVR1, TGFBR3), transcription
factors (MYCN, possibly CCDC148), and ubiquitin-proteasome
(RFWD2, FANCL, CACYBP) proteins that may interact to
modulate cell signaling. The Acvr1 SNP rs6406107 eliminates a
putative transcription factor binding site and diminished DNA–
protein binding making this gene worthy of future investigations
in acute lung injury.
Author Disclosure: G.D.L. is employed by the University of Pittsburgh. V.J.C. does
not have a financial relationship with a commercial entity that has an interest in
the subject of this manuscript. P.L. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. K.B.
does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. A.B. does not have a financial relation-
ship with a commercial entity that has an interest in the subject of this
manuscript. K.G. does not have a financial relationship with a commercial entity
that has an interest in the subject of this manuscript. A.S.J. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. K.A.B. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. M.D. is employed by
the University of Cincinnati. H.P.-V. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. R.A.D.
does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. Y.P.P.D. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
manuscript. Q.L. does not have a financial relationship with a commercial entity
that has an interest in the subject of this manuscript. L.J.V. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. M.M. is employed by the University of Cincinnati. N.K. was
a consultant for Sanofi-Aventis and Stromedix. He received lecture fees form
Medimmune and owns a patent on use of microRNAs to diagnose and treat IPF
Us or peripheral blood biomarkers to diagnose and predict outcome in IPF. M.Y.
is employed by the Medical College of Wisconsin. D.R.P. does not have a financial
relationship with a commercial entity that has an interest in the subject of this
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dbSNPSNP TypePhenotype (%) SymbolDescription ChrStart Position
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