Bleomycin-induced pulmonary fibrosis susceptibility genes in AcB/BcA recombinant congenic mice

Anne-Marie Lemay and Christina K. Haston

Department of Human Genetics and the Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The genetic basis of susceptibility to pulmonary fibrosis is largely unknown. Initially, in this study, loci regulating the response of bleomycin-induced pulmonary fibrosis were mapped using a set of recombinant congenic strains bred from pulmonary fibrosis-resistant A/J and susceptible C57BL/6J (B6) mice. Linkage was identified (logarithm of the odds score = 4.9) on chromosome 9, and other suggestive loci were detected. The putative loci included alleles from both the B6 and A/J strains as increasing the fibrosis response of congenic mice. Gene expression analysis with microarrays revealed 3,304 genes or expressed sequence tags to be differentially expressed (P < 0.01) in lung tissue between bleomycin-treated B6 and A/J mice, and 246 of these genes mapped to potential susceptibility loci. Pulmonary genes differentially expressed between bleomycin-treated B6 and A/J mice included those of heparin binding and extracellular matrix deposition pathways. A review of available genomic sequences revealed 809 (43% of total) genes in the linkage intervals to have variations predicted to alter the encoded proteins or their regulation, 68 (8.4%) of which were also differentially expressed. Genomic approaches were combined to produce a set of candidate genes that may influence susceptibility to bleomycin-induced pulmonary fibrosis in the A/J:B6 mouse model.

genetic predisposition to disease; quantitative trait loci; microarray analysis; recombinant congenic strains


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PULMONARY FIBROSIS is a genetically complex disease that can result from known exposures such as chemotherapy regimens involving bleomycin or can occur idiopathically (11, 21). The pathology of excessive deposition of the extracellular matrix in the lung interstitium can result in impaired lung function and, ultimately, respiratory failure. From clinical studies, it is suggested that the development of pulmonary fibrosis has a genetic component (2, 28), but the specific genes involved have not been identified.

Inbred strains of mice differ in their tendency to develop pulmonary fibrosis after bleomycin treatment, and they have been used as the base of genetic investigations to define susceptibility genes. We have previously mapped two quantitative trait loci (QTL), named bleomycin-induced pulmonary fibrosis 1 and 2 (Blmpf1 and Blmpf2), of the propensity to develop fibrosis after bleomycin exposure in F2 mice derived from progenitor strains C57BL/6J (B6) and C3Hf/KAM. Barth et al. (1) used the progenitor strains DBA/2 and BALB/c to map two loci of susceptibility to bleomycin-induced pulmonary fibrosis, which, as they differ from those we have mapped (15), indicates the utility of studies in distinct inbred strains of mice for uncovering susceptibility loci of complex traits.

In this study, we made use of the strain difference in the bleomycin response between B6 (susceptible) and A/J (resistant) mice (31) and of available genomic resources to both map susceptibility to pulmonary fibrosis and to identify a set of potential candidate genes for the trait. Investigations of the A/J strain were undertaken as our previously identified locus Blmpf1, which maps to the major histocompatibility complex (MHC), was defined in B6 (MHC haplotype H2b) and C3Hf/KAM (H2k) mice, and, as the haplotype of the A/J strain (H2a) is different, studies of this strain may reduce the number of MHC-derived fibrosis candidate genes.

To map the susceptibility to pulmonary fibrosis, we used a series of 36 recombinant congenic mouse strains (RCS) derived from B6 and A/J progenitor strains (7, 8). Fourteen of the strains (named AcB) contain a random 13.25% of B6 genes in the A/J strain background, and 22 strains are 13.25% A/J genes in the B6 background (BcA strains). Thus, with this resource, B6 alleles involved in the susceptibility to pulmonary fibrosis are potentially divided among 14 strains of mice, enabling the assessment of the effect of discrete B6 genomic regions on the phenotype. Such RCSs have been used by others to map complex traits of malaria susceptibility and endotoxin-induced lung response among others (5, 6, 8 ).

In addition, among the mapped positional candidates, a set of potential fibrosis susceptibility genes was isolated by identifying the subset of these genes that were differentially expressed between B6 and A/J mice in bleomycin-treated mouse lungs and for which there is a sequence variation between the two strains.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
Mice of the C57BL/6J and A/J strains were purchased from the Jackson Laboratory and mice of the AcB/BcA strains from Emerillon Therapeutics (Montreal, Quebec, Canada). The AcB and BcA series of mouse RCS were generated and maintained at the Montreal General Hospital Research Institute according to a breeding scheme and a genotyping protocol previously described (7). Male and female mice of 8–14 wk of age were used for the study. All mice were handled according to protocols approved by McGill University Animal Care Committee and the guidelines and regulations of the Canadian Council on Animal Care.

Bleomycin treatment.
Lung damage was elicited by administering bleomycin through osmotic minipumps implanted subcutaneously, as described previously (15). A/J mice were typed for their fibrosis response at 3 wk (5 males and 6 females) or 6 wk (6 males and 6 females) after treatment, and 10 untreated control mice (5 males and 5 females) were killed at the 6-wk time point. Male mice received 100 U bleomycin/kg body wt (~2.5 U/mouse), and female mice received 125 U/kg. Male and female mice were treated in separate studies due to the higher drug dose required to produce fibrosis in female mice. An additional 11 A/J and 7 B6 mice were treated with a lower dose of bleomycin (80 U/kg for males and 100 U/kg for females) and killed after 3 wk; 208 RCS mice (a minimum of 3 male and 3 female mice of each of 19 RCS, and a total of 3–5 male and female mice for 14 additional strains) received 80 U/kg for males and 100 U/kg for females to assay the fibrosis response. A further 8–15 mice of each of the AcB65 and the BcA 70, 72, 78, 81, 84, and 85 strains were treated with the lower-dose bleomycin protocol to substantiate their phenotypes. AcB/BcA mice were killed at 3 wk after treatment. AcB52, AcB56, and BcA76 strains were not studied due to low availability of these strains.

Histology and fibrosis scoring.
At autopsy, the lungs were removed, and the single left lobe of each mouse was perfused with 10% neutral buffered formalin and submitted for histological processing. Lung sections were stained with Masson’s trichrome to identify the sites of collagen deposition in the lung. The area of the fibrosing phenotype for each mouse was quantified with image analysis of histological sections as in our previous study (15). Specifically, the area of fibrosis in the left lung lobe was determined from a user-drawn region surrounding the fibrosis (Spot Software) and compared with the area of the entire lobe to yield the percent pulmonary fibrosis for individual mice. Two different users evaluated the percent fibrosis of the mice, and the interuser agreement was r2 = 0.87.

QTL analysis.
Genome scan analyses were performed by using MapManager QTX (version b20) (27). With this software, the set of fibrotic phenotypes (defined as the percentage of the lung with fibrosis by histology) of the 33 RCS mice was compared with their known genotypes to identify the genetic loci influencing this trait in B6 versus A/J mice. Only the RCSs with three or more phenotyped mice were included in the QTL analysis (this yielded 33 strains when both sexes were combined, 27 strains for males only, and 23 strains for females only). In this analysis, marker regression function was used to determine the likelihood ratio statistic for each of 616 markers on 20 chromosomes. For each marker, the resultant likelihood ratio statistic was divided by 4.61 (2 x ln 10) to yield the logarithm of the odds (LOD) score. The thresholds for determining the significance of loci were based on Lander and Kruglyak (22)-proposed linkage standards and on empirically derived limits. From Lander and Kruglyak, we used the mouse backcross value (deemed closest to recombinant congenic mice), which is a suggestive linkage LOD score of 1.9 and a significant LOD score of 3.3. To empirically determine suggestive and significant threshold LOD scores, 10,000 permutations of the phenotype on the genotype were carried out in our data set. With the use of the data of 33 RCSs, the LOD score suggestive of linkage was 1.8, for significant linkage the LOD score was 3.8, and the LOD score indicative of highly significant linkage was 6.9.

Gene expression.
After death, the right lung of each mouse was immediately homogenized in 2 ml TRIzol reagent and placed in dry ice. The homogenates were stored at –85°C until RNA isolation. Total RNA was extracted from A/J lung homogenates according to the manufacturer’s (Sigma) instructions. The RNA from the right lungs of four or five mice from each group, defined by sex and treatment, was pooled as in Ref. 30 to minimize biological variation in gene expression within a group. One sample of pooled RNA for each group was processed through a RNAEasy column (Qiagen) and submitted for hybridization. The quality of the isolated RNA was assessed and confirmed both before and after pooling by using the Agilent Bioanalyzer (Agilent Technologies; Palo Alto, CA). The experiment was performed with 1 chip/mouse group, represented by its pooled RNA. The gene expression profile of the following groups of A/J mice was measured at the 3-wk time point: males, 100 U bleomycin/kg; females, 125 U/kg; males, 80 U/kg; females, 100 U/kg; and male untreated control mice and female untreated control mice. The gene expression profile of the following groups of mice was measured at the 6-wk time point: males, 100 U/kg; and females, 125 U/kg.

Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at the McGill University and Genome Quebec Innovation Centre. Probe synthesis, hybridization, and washing protocols followed the standardized Affymetrix protocol as reported by Novak et al. (29).

The chips were scanned with a GeneArray Scanner (Agilent Technologies). The resultant gene expression profile was then viewed using Microarray Suite 5.0 (Affymetrix). MOE430A GeneChip arrays containing 22,690 probe sets derived from sequence clusters contained in Build 107, June 2002, of UniGene, which represent 12,422 functionally annotated genes and a set of expressed sequence tags, were used.

Microarray data analysis.
Routines from Bioconductor version 1.4 (http://www.bioconductor.org/) within the R version 1.90 statistical language (17) were used for quality control, normalization, and differential expression. In particular, the quality of the raw microarray data was assessed by inspecting similarities between the intensity distribution and RNA digestion plot for each array. Normalization was performed using the robust probe level model (18). Using mean log intensity versus average log intensity plots, we compared arrays to determine whether different times of postbleomycin exposure, bleomycin dose, and/or gender of the animal influenced gene expression in A/J mice, and we found no significant differences in expression levels, with the exception of the genes on the X and Y chromosomes. This lack of difference justified the pooling of data from these arrays to form two distinct groups: A/J control and A/J bleomycin. A list of significantly differentially expressed genes with P < 0.01 was then generated intrastrain (control vs. bleomycin exposure) with the detection of differential expression performed using the LIMMA package (25, 33). The gene expression data of A/J mice were then compared, using LIMMA analysis, with those reported for B6 mice in response to the same bleomycin treatment (16).

To further assess the lung gene expression profile of A/J mice in response to bleomycin, we used the LIMMA package to analyze the data from NCBI GEO entry GDS350. These data were generated from four Gladstone v2 mouse lung oligo arrays (n = 16,463 probes) hybridized with cDNA from each of four bleomycin-treated A/J mice compared with the pooled RNA of control untreated A/J mice.

The detection of significantly overrepresented Gene Ontology categories was performed using the GOStats package in bioconductor (10). This test of statistical significance considers the number of differentially expressed genes found in each category compared with the total number of genes in the category represented on the chip.

Quantitative real-time PCR.
Four to five micrograms of total RNA from each of four mice of each treatment group were used in a RT reaction to synthesize first-strand cDNA using oligo(dT)12–18 Primer and Superscript II RNase H- Reverse Transcriptase (Invitrogen; Carlsbad, CA) in a 20-µl total volume. The lung expression level of each of the genes in Table 1 was determined for bleomycin-treated mice (100 U/kg for female and 80 U/kg for males at the 3-wk time point) and control B6 and A/J mice. These six genes were selected to represent genes of increased and decreased expression in response to bleomycin, as indicated by the arrays. For this analysis, sequence-specific primer sets were designed using Primer 3 (32) or taken from Primerbank (35). Primers were selected to span large introns to amplify only cDNA (see Table 1).


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Table 1. Genes investigated with real-time PCR

 
For real-time PCR, Qiagen SYBR green and a LightCycler (Roche) were used. Each reaction contained 1 µl of cDNA template and 10 µl of the QuantiTect SYBR green PCR Kit (Qiagen). The PCR variables were as follows: 95°C for 15 s, 55°C for 20 s, and 72°C for 20 s repeated for 50 cycles. Genomic DNA, no reverse-transcribed RNA, and no template controls were included in the runs. Relative gene expression data analysis was carried out with the standard curve method (36). The fluorescence data were expressed as normalized to a reference gene, Spinocerebellar ataxia 10 homolog (human) Sca10, which was determined with the array data to be of invariant expression across treatment types.

Applied Biosystems Real-Time PCR system 7500 was used with the Taqman gene expression assay to test fibroblast growth factor receptor 2 expression. In this assay, each 25-µl reaction contained 1 µl of 1:10 diluted cDNA template, 12.5 µl of TaqMan Universal PCR Master Mix, and 1.25 µl of Assays-on-Demand Gene Expression Assay Mix, which contained forward and reverse primers and labeled probe. The default thermal cycling conditions for PCR were used as instructed by the manufacturer. Relative quantification values were obtained by using Applied Biosystems software and Sca10 reference gene expression.

Sequence comparison.
The markers flanking each of the identified putative loci were located in the Celera (Rockville, MD) mouse genome database (http://www.celera.com/, CDS 13h release), and the number of genes (excluding pseudogenes) mapping to each region was determined. With the use of DNA positions of the flanking markers, the Celera Mouse single nucleotide polymorphism (SNP) reference database (version 3.6) was queried for SNPs within each linkage region. These SNP data were then filtered to uncover the set of SNPs for which B6 and A/J mice have a different allele. These data were further filtered to exclude SNPs appearing in the intronic region or identified as synonymous, as has been used by others (24).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fibrosis phenotype of A/J and B6 mice.
To enable the use of AcB/BcA recombinant congenic mice in mapping susceptibility to bleomycin-induced pulmonary fibrosis, the response of the A/J strain to the drug (delivered by osmotic minipumps) relative to the known response of B6 mice (16) was determined. Male and female A/J mice were treated with 100 and 125 U bleomycin/kg, respectively, and groups of mice of each sex were killed 3 or 6 wk later. The A/J phenotype of the percentage of the lung with fibrosis, as assessed with histology, was similar at 3 and 6 wk (0.53% at 3 wk and 0.49% at 6 wk, P = 0.41) and did not differ by sex (P = 0.13, data not shown). The amount of fibrosis in A/J mice was lower than that reported for the B6 strain for comparisons in both male (B6 fibrosis = 11.5 ± 5.5%, P = 4.4 x 10–6) and female mice (B6 fibrosis = 7.8 ± 3.9%, P = 3.0 x 10–6), as reported in Haston et al. (16).

Because of the risk of lethality from acute toxicity in response to bleomycin, which is not related to the development of fibrosis, we assayed the lung phenotype of B6 and A/J mice 3 wk after delivery of a lower dose of bleomycin (80 U/kg for males and 100 U/kg for females). As the strain difference in fibrosis phenotype of B6 and A/J mice was evident at this dose in both male (B6 = 5.7 ± 1.1% vs. A/J = 0.12 ± 0.18%, P = 1 x 10–7) and female mice (B6 = 4.7 ± 1.9% vs. A/J = 0.48 ± 0.56%, P = 4.7 x 10–4), this lower dose was used in the mapping study.

AcB/BcA fibrosis phenotype.
The first step of our strategy for identifying the genes involved in the fibrosis susceptibility of B6 mice relative to the A/J strain was to determine their map position. To accomplish this, we treated a minimum of 3 male and 3 female mice of 19 AcB/BcA strains and 3–5 mice of additional 14 RCS with bleomycin and killed the mice 3 wk later. The percent fibrosis of the lung, by histology, was used to phenotype for susceptibility/resistance, and the resultant strain distribution pattern of the RCS mice is shown in Fig. 1. As shown in Fig. 1, mice of the BcA strains were generally more sensitive to the development of fibrosis than those of the AcB strains. Two BcA strains (BcA 78 and 81) were highly susceptible to the development of pulmonary fibrosis. These strains had an average of 10% and 15% fibrotic lung tissue, which is 1.8 and 2.7 times the B6-susceptible phenotype at this dose. In addition, BcA strains 68, 69, 73, 74, 79 (females only), 84, and 85 had significantly lower levels of fibrosis compared with B6 mice (<1% fibrosis, all P < 8.6 x 10–3).



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Fig. 1. Strain distribution pattern of the bleomycin-induced pulmonary fibrosis response of recombinant congenic mice. The average percent fibrosis (±SE), measured from histological lung sections of mice 3 wk after a dose of 80 [males (m)] or 100 U bleomycin/kg [females (f)], is given. The number of phenotyped mice of each strain is indicated below the strain names. Six strains demonstrated a sex-specific response, and the data are presented separately. B6, C57BL/6J mice.

 
As expected from their A/J background, 9 of 12 AcB strains were of a fibrosis-resistant phenotype, like A/J mice, and the other 3 AcB strains showed intermediate susceptibility (percent fibrosis significantly higher than that of A/J mice and significantly lower than that of B6, all P < 0.03; see Fig. 1).

Mapping of pulmonary fibrosis susceptibility.
With the use of percent fibrosis as a quantitative phenotypic trait and the genotypic data of all 33 phenotyped AcB and BcA strains combined, 2 linkage regions were detected through linear regression analysis with MapManager QTX software (27) (see Table 2). The identified linkages (on chromosomes 3 and 6) have suggestive LOD scores (22), and they are also suggestively linked to the phenotype according to the permutation test in MapManager QTX. When regression was performed using the dataset from male mice only (n = 27 strains with 3 or more phenotyped male mice), suggestive regions on chromosomes 1, 5, 9, and 12 were detected in addition to the regions on chromosomes 3 and 6. No regions suggested to be linked to the fibrosis phenotype were evident with the data from the female mice (23 strains with 3 or more phenotyped male mice) alone. The difference in linkage results between the sexes is likely attributable to the lower number of phenotyped recombinant congenic female mice compared with males and to the greater range of phenotype in male mice. At each of these putative loci, the presence of B6 alleles increased the fibrotic phenotype of RCS mice, as shown in Table 2.


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Table 2. Putative linkage regions: effect of genotype on bleomycin-induced pulmonary fibrosis phenotype of RCS mice

 
When the 21 BcA strains were analyzed separately, QTL were identified on chromosomes 9, 15, and 18 (LOD scores between 2.0 and 4.9; see Table 2). At each of these putative loci, the presence of A/J alleles increased the fibrotic phenotype of RCS mice, as shown in Table 2. This may indicate that A/J loci could combine with B6 loci to increase the pulmonary fibrosis to a level exceeding the parental fibrosis levels. No loci were found through analysis of the AcB strains separately, which is likely due to the smaller range of the phenotype in these strains. The percentage of the phenotype explained by each of the loci was between 35% and 72% based on regression analysis in MapManager QTX. As this was calculated from the differences between two homozygous populations at each marker, the phenotypic variance results are of limited usefulness.

Candidate gene identification.
To propose candidate fibrosis susceptibility genes, we isolated, from among the mapped positional candidates (identified by a composite of Ensembl and Celera), the subset of genes that were differentially expressed between B6 and A/J mice in bleomycin-treated lungs and for which there was a sequence variation between the strains.

Gene expression studies.
It was hypothesized that the genes producing the difference in response to bleomycin between B6 and A/J mice would be differentially expressed in the lungs of these mice after the drug treatment. To identify such genes, expression studies were performed using Affymetrix GeneChip microarrays. The lung response of A/J mice to the drug was ascertained by comparing the gene expression profile of bleomycin-treated mice with that of untreated controls, and, subsequently, this dataset was compared with that of B6 mice (16) to identify strain differences in the response.

The A/J response to bleomycin was measured with six arrays, each of which represents the response of a group of treated mice, as described in MATERIALS AND METHODS, compared with two arrays of gene expression in lung tissue from untreated mice. Nine genes were identified to be differentially expressed (fold ≥ 2, P < 0.01, with a maximum fold change = 5.8, P = 0.0003) between control and bleomycin-treated A/J mice (see Table 3). This limited change in gene expression reflects the minimal histological response of A/J mice to bleomycin, which was consistent across the groups of A/J mice evaluated. In support of this finding, we analyzed the data taken from NCBI entry GEO GDS350 of the pulmonary response to bleomycin of A/J mice, and we detected no differentially expressed genes for bleomycin to control comparison (all genes P ≥ 0.14). In contrast to the minimal bleomycin response of A/J mice, the B6 gene expression profile, as reported in Haston et al. (16), has 1,768 genes or expressed sequence tags measured to be differentially expressed between controls and bleomycin-treated mice.


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Table 3. Significantly differently expressed genes in the lungs of bleomycin-treated A/J mice relative to untreated A/J mice

 
Next, we compared the gene expression profile of A/J mice with that of B6 mice to identify strain differences in lung gene expression for both untreated control mice (two B6 arrays compared with two A/J arrays) and in response to bleomycin (five B6 arrays compared with six A/J arrays); 357 genes were differentially expressed (P < 0.01) in the lungs of untreated A/J mice compared with B6 control mice, whereas 2,555 genes and 749 expressed sequence tags were differentially expressed between the strains after bleomycin treatment (P < 0.01). A majority (90%) of the genes of altered expression in the controls were also present in the treated comparison, and 55% of the differentially expressed genes were more highly expressed in lungs of A/J mice. To assess the validity of the microarray data, six genes were chosen from the list of B6:A/J differentially expressed genes and were submitted for RT-PCR analysis. As shown in Fig. 2, the expression levels measured with RT-PCR were found to be similar to the microarray results.



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Fig. 2. Comparison of microarray gene expression data with real-time PCR evaluation of selected genes. Shown if the fold change (±SE) in lung expression of untreated control (ctrl) A/J mice relative to B6 and for bleomycin (bleo)-treated mice. Cyp4b1, cytochrome P-450, family 4, subfamily b, polypeptide 1 gene; Ltbp2, latent transforming growth factor-ß binding protein 2 gene; Dpep1, dipeptidase 1 gene; Pten, phosphatase and tensin homolog gene; Fgfr2, fibroblast growth factor receptor 2 gene; Glo1, glyoxalase 1 gene.

 
The 3,304 genes or expressed sequence tags identified to be differentially expressed in lung tissue between B6 and A/J bleomycin-treated mice are given in Supplemental Table S1 (available at the Physiological Genomics web site).1 To present the cellular processes represented in this set of genes, GOStats from Bioconductor (10) was used to compare the gene ontology distribution of these genes with that of all probes present on the microarray chip. By this analysis, the genes differentially expressed in the lungs between bleomycin-treated A/J and B6 mice were related to heparin binding (P = 2.7 x 10–7), glutathione transferase activity (P = 1.6 x 10–6), and extracellular matrix structural constituents (P = 1.3 x 10–4). The heparin binding category includes genes such as fibronectin 1, tenascin XB, fibroblast growth factor 1 (Fgf1), and thrombospondin 1. The glutathione transferase activity category includes glutathione S-transferases, and extracellular matrix structural constituents were mainly procollagens and laminin.

The map positions of the genes measured to be differentially expressed in the lungs of bleomycin-treated B6 mice compared with A/J mice were reviewed to isolate genes located in the putative linkage regions. Two hundred forty-six linkage interval genes were identified to be differentially expressed (P ≤ 0.01), and the subset of 18 of these genes, with fold changes in expression ≥2, is shown in Table 4. These genes are considered to be expression and positional candidates for the genetic basis of bleomycin-induced pulmonary fibrosis in this model.


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Table 4. A/J:B6 differentially expressed genes of the linkage regions

 
Sequence variation.
It was hypothesized that the gene (or the regulatory region of the gene) producing the difference in response to bleomycin between B6 and A/J mice would be polymorphic between these two strains. Thus we documented the sequence variation between these strains for the putative linkage regions. These data were generated through use of the Celera Discovery System and Celera’s associated databases. Among the 1,899 genes located in the loci, 809 genes had B6:A/J polymorphisms in their coding sequences or untranslated regions. In the 809 genes containing SNPs, 68 genes were also differentially expressed (P ≤ 0.01) and are thus positional, expression- and sequence-based candidate genes for the B6 versus A/J difference in susceptibility to bleomycin-induced pulmonary fibrosis. Five of the linkage region genes with expression fold change ≥2 have SNPs (see Table 4).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A combination of mapping, gene expression, and DNA sequence analysis was used to identify specific candidate genes of susceptibility to bleomycin-induced pulmonary fibrosis in an A/J:B6 mouse cross. The candidate genes are of extracellular matrix deposition and immune response pathways and may represent new players in the complex pathology of pulmonary fibrosis.

Using the osmotic minipump delivery method, we showed the mice of the A/J strain to have a minimal fibrotic response to bleomycin, in contrast to the inflammation and fibrosis that develops in B6 mice (13, 31). This mode of bleomycin delivery, developed by Harrison and Lazo (12) and used by us (15), was selected as it has been found to produce a fibrotic phenotype that more closely resembles idiopathic pulmonary fibrosis than the more commonly used experimental method of intratracheal drug delivery (9). The response of the A/J strain to the drug agrees with the findings of Rossi et al. (31), in which bleomycin was delivered intraperitoneally to mice over 4 wk and fibrosis did not result, but differs from the report of Chen et al. (3), in which an intratracheal drug delivery system was used.

With the confirmed strain difference in bleomycin-induced fibrosis susceptibility, recombinant congenic mice were used to map nine loci of the phenotype. Two of the putative linkage regions, on chromosomes 6 and 18, overlap with previously defined QTL of susceptibility to radiation-induced pulmonary fibrosis (14), and the locus on chromosome 6 may coincide with a QTL of bleomycin-induced pulmonary fibrosis that has been reported to be on this chromosome (1). The commonality of the loci supports their existence, but as the present linkage regions were mapped in a limited number of RCSs, confirmatory studies are required. If confirmed, the implication of the same linkage regions in susceptibility to both radiation-induced and bleomycin-induced pulmonary fibrosis may indicate that the phenotype causative genes underlying these loci are not specific to the damaging agent used but are related to the development of fibrosis. In addition, the putative fibrosis linkage region indicated by the marker D3Mit335 overlaps a butylated hydroxytoluene-induced inflammation (lymphocytes) QTL (26), which may indicate this to be a common lung response locus. The linkage regions of bleomycin-induced pulmonary fibrosis susceptibility detected in a B6 x C3Hf/KAM cross did not meet the criterion for suggestive linkage with the present data set; the LOD score of Blmpf1 was 1.7, and the LOD score of Blmpf2 was 1.5.

Our second genomic approach for identifying fibrosis susceptibility genes was to measure the gene expression profile of A/J mouse lungs after drug treatment and compare it with that documented for B6 mice. The B6 gene expression data, reported in Haston et al. (16) and used for comparison in the present investigation, agree with those reported in previous studies of this strain (19, 20) and include the representation of 41 bleomycin-induced differentially expressed B6 genes in a gene cluster (n = 66 genes) defined by Kaminski et al. (19) for fibrosis development. The A/J response to bleomycin, measured by pulmonary gene expression of phenotypically similar groups, also agreed with a separate report for this strain (NCBI GEO). The comparison of the gene expression profile of A/J and B6 mice revealed thousands of genes to be differentially expressed by strain, further indicating the complexity of the fibrosis phenotype.

By combining the genomic approaches of linkage and gene expression (as in Ref. 23), fibrosis-causative candidate genes for the B6:A/J model were proposed. The identified genes are considered candidates as the causal variation leading to the development of bleomycin-induced pulmonary fibrosis with the assumption that the fibrosis-causative gene is differentially expressed in the bleomycin-treated lung. Differential expression is not a necessary condition for implication as causal variation but was used to rank the set of positional candidate genes to facilitate further investigation. From this analysis, the possible pathways to fibrosis in the A/J:B6 model include differences in immune system mediators and in extracellular matrix homeostasis. Specifically, gene candidates from the linkage regions that showed an increase in expression in the lungs of A/J mice compared with B6 mice are involved in immune defense, such as the immunoglobulin heavy chains 1 and 4 and the J558 family, whereas genes such as procollagen 3{alpha}1 and 5{alpha}2 and elastin, which are linked to the collagen deposition and turnover, were of relatively increased expression in the lungs of B6 mice. Second, from the analysis of the 21 BcA strains, we were able to detect loci where alleles from the resistant A/J strain increased the fibrotic phenotype of BcA mice, which likely indicates an interaction among loci influences the development of fibrosis. As an example of such an interaction, Fgf1, which maps to a locus where A/J alleles increase the phenotype, showed an increase in expression in the lungs of A/J mice relative to B6, and it could interact with fibroblast growth factor receptor 1 (Fgfr1), which was shown to be increased in B6 mice, to produce increased levels of pulmonary fibrosis in certain BcA strains.

We also assessed the positional candidate genes for DNA sequence variation and, as with gene expression, the functional affect of any sequence variation would have to be confirmed but the existence of a coding or regulatory SNP is potential supporting evidence for causal variation. The Celera database was used as the source of SNPs as it is the most complete documentation of the A/J strain at present, although it may not be a comprehensive review of all B6:A/J sequence variation. With this analysis, a finite set of sequence variation in the candidate genes (shown in Table 4) was uncovered for further testing. Included in this list are the physiological candidate genes lysyl oxidase and interleukin-1 receptor-associated kinase 4. Lysyl oxidase is involved in the cross-linking of collagen, and persistent expression of the gene has been implicated in irreversible fibrosis in bronchiolitis obliterans (34), whereas interleukin-1 receptor expression has been shown to increase with the development of fibrosis in B6 mice (3).

In summary, a combination of genomic approaches was used to identify candidate genes for susceptibility to bleomycin-induced pulmonary fibrosis in a B6:A/J mouse cross. Thirty-three recombinant congenic mouse strains were used to define six intervals, which ranged in size between 9.4 and 29.8 Mb, linked to the trait. Three more putative loci were defined using 21 BcA strains only, and these contain alleles where the A/J genotype increases the fibrosis phenotype. In addition, gene expression studies identified a set of differentially expressed genes mapping to these nine intervals, and a review of SNP data permitted the identification of parental strain gene sequence variations.

The phenotypic trait mapped, susceptibility to bleomycin-induced pulmonary fibrosis, is clinically significant as this lung response limits the dose of bleomycin that can be safely administered and as the induced injury may be a model for the more prevalent condition of idiopathic pulmonary fibrosis. The specific genetic variants reported, if confirmed to influence drug-induced pulmonary fibrosis, could provide insight on the development of this pathology.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by funding from the Canadian Institutes of Health Research and Fonds de la Recherche en Santé Québec.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. K. Haston, Dept. of Human Genetics and the Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain, Montreal, Quebec, Canada H2X 2P2 (e-mail: christina.haston{at}mcgill.ca).

1 The Supplemental Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00095.2005/DC1. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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