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Molecular subsets in the gene expression signatures of scleroderma skin.

Milano A, Pendergrass SA, Sargent JL, George LK, McCalmont TH, Connolly MK, Whitfield ML - PLoS ONE (2008)

Bottom Line: The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings.Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Dartmouth Medical School, Hanover, New Hampshire, United States of America.

ABSTRACT

Background: Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.

Methodology and findings: We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.

Conclusions and significance: Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

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Related in: MedlinePlus

Gene expression signatures in scleroderma.4,149 probes that changed at least 2-fold from their median value on at least two microarrays were selected from 75 microarray hybridizations representing 61 biopsies. Probes and microarrays were ordered by 2-dimensional average linkage hierarchical clustering. This clustering shows that the dSSc, lSSc, morphea samples form distinct groups largely stratified by their clinical diagnosis. A. The unsupervised hierarchical clustering dendrogram shows the relationship among the samples using this list of 4,149 probes. Samples names have been color-coded by their clinical diagnosis: dSSc in red, lSSc in orange, morphea and EF in black, and healthy controls (Nor) in green. Forearm (FA) and Back (B) are indicated for each sample. Solid arrows indicate the 14 of 22 forearm-back pairs that cluster next to one another; dashed arrows indicate the additional 3 forearm-back pairs that cluster with only a single sample between them. Technical replicates are indicated by the labels (a), (b) or (c). 9 out of 14 technical replicates cluster immediately beside one another. B. Overview of the gene expression profiles for the 4,149 probes. Each probe has been centered on its median expression value across all samples analyzed. Measurements that are above the median are colored red and those below the median are colored green. The intensity of the color is directly proportional to the fold change. Groups of genes on the right hand side indicated with colored bars are shown in greater detail in panels C–H. C. Immunoglobulin genes expressed highly in a subset of patients with dSSc and in patients with morphea, D. proliferation signature, E. collagen and extracelluar matrix components, F. genes typically associated with the presence of T-lymphocyes and macrophages, G. Genes showing low expression in dSSc, H. Heterogeneous expression cluster that is high in lSSc and a subset of dSSc. In each case only a subset of the genes in each cluster are shown. The precise location of each gene in the cluster can be viewed in Supplemental Figure S1.
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pone-0002696-g001: Gene expression signatures in scleroderma.4,149 probes that changed at least 2-fold from their median value on at least two microarrays were selected from 75 microarray hybridizations representing 61 biopsies. Probes and microarrays were ordered by 2-dimensional average linkage hierarchical clustering. This clustering shows that the dSSc, lSSc, morphea samples form distinct groups largely stratified by their clinical diagnosis. A. The unsupervised hierarchical clustering dendrogram shows the relationship among the samples using this list of 4,149 probes. Samples names have been color-coded by their clinical diagnosis: dSSc in red, lSSc in orange, morphea and EF in black, and healthy controls (Nor) in green. Forearm (FA) and Back (B) are indicated for each sample. Solid arrows indicate the 14 of 22 forearm-back pairs that cluster next to one another; dashed arrows indicate the additional 3 forearm-back pairs that cluster with only a single sample between them. Technical replicates are indicated by the labels (a), (b) or (c). 9 out of 14 technical replicates cluster immediately beside one another. B. Overview of the gene expression profiles for the 4,149 probes. Each probe has been centered on its median expression value across all samples analyzed. Measurements that are above the median are colored red and those below the median are colored green. The intensity of the color is directly proportional to the fold change. Groups of genes on the right hand side indicated with colored bars are shown in greater detail in panels C–H. C. Immunoglobulin genes expressed highly in a subset of patients with dSSc and in patients with morphea, D. proliferation signature, E. collagen and extracelluar matrix components, F. genes typically associated with the presence of T-lymphocyes and macrophages, G. Genes showing low expression in dSSc, H. Heterogeneous expression cluster that is high in lSSc and a subset of dSSc. In each case only a subset of the genes in each cluster are shown. The precise location of each gene in the cluster can be viewed in Supplemental Figure S1.

Mentions: We identified 4,149 probes whose expression varied from their median values in these samples by more than 2-fold in at least two of the 75 arrays and analyzed them by two-dimensional hierarchical clustering [18]. The resulting sample dendrogram shows that the samples separate into two main branches (Figure 1A) that in part stratify patients by their clinical diagnosis. The branch lengths in the tree are inversely proportional to the correlation between samples or groups of samples. The diversity in gene expression among the patients with scleroderma is greater than previously shown (Figure 1B) [16], [17] as distinct subsets of scleroderma are evident in the gene expression patterns. Some of these delineate existing classifications, such as the distinction between limited and diffuse, while others reflect new groups. One subset of dSSc patients cluster on the left branch (red) and has gene expression profiles that are distinct from both healthy controls and patients with lSSc (Figure 1B–1C), while a second subset of dSSc skin clusters in the middle of the dendrogram tree (black), and a third set clusters with healthy controls. We found lSSc samples formed a group in the middle portion of the dendrogram and could be associated with a distinct, but heterogeneous gene expression signature that also showed high expression in a subset of dSSc patients (Figure 1H). LSSc samples are partially intermixed with normal controls on the right boundary and with dSSc on the left boundary of the tree, illustrating that their gene expression phenotype is highly variable (Figure 1A). Samples taken from individuals with morphea also grouped together with a gene expression signatures that overlapped with those of dSSc and lSSc (Figure 1). Although nodes can be flipped, we have left the nodes of the dendrogram as originally organized by the clustering software, which places nodes with the most similar samples next to one another. Although, the assignment of samples into particular clusters (Table 3) would not change even if nodes were flipped.


Molecular subsets in the gene expression signatures of scleroderma skin.

Milano A, Pendergrass SA, Sargent JL, George LK, McCalmont TH, Connolly MK, Whitfield ML - PLoS ONE (2008)

Gene expression signatures in scleroderma.4,149 probes that changed at least 2-fold from their median value on at least two microarrays were selected from 75 microarray hybridizations representing 61 biopsies. Probes and microarrays were ordered by 2-dimensional average linkage hierarchical clustering. This clustering shows that the dSSc, lSSc, morphea samples form distinct groups largely stratified by their clinical diagnosis. A. The unsupervised hierarchical clustering dendrogram shows the relationship among the samples using this list of 4,149 probes. Samples names have been color-coded by their clinical diagnosis: dSSc in red, lSSc in orange, morphea and EF in black, and healthy controls (Nor) in green. Forearm (FA) and Back (B) are indicated for each sample. Solid arrows indicate the 14 of 22 forearm-back pairs that cluster next to one another; dashed arrows indicate the additional 3 forearm-back pairs that cluster with only a single sample between them. Technical replicates are indicated by the labels (a), (b) or (c). 9 out of 14 technical replicates cluster immediately beside one another. B. Overview of the gene expression profiles for the 4,149 probes. Each probe has been centered on its median expression value across all samples analyzed. Measurements that are above the median are colored red and those below the median are colored green. The intensity of the color is directly proportional to the fold change. Groups of genes on the right hand side indicated with colored bars are shown in greater detail in panels C–H. C. Immunoglobulin genes expressed highly in a subset of patients with dSSc and in patients with morphea, D. proliferation signature, E. collagen and extracelluar matrix components, F. genes typically associated with the presence of T-lymphocyes and macrophages, G. Genes showing low expression in dSSc, H. Heterogeneous expression cluster that is high in lSSc and a subset of dSSc. In each case only a subset of the genes in each cluster are shown. The precise location of each gene in the cluster can be viewed in Supplemental Figure S1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481301&req=5

pone-0002696-g001: Gene expression signatures in scleroderma.4,149 probes that changed at least 2-fold from their median value on at least two microarrays were selected from 75 microarray hybridizations representing 61 biopsies. Probes and microarrays were ordered by 2-dimensional average linkage hierarchical clustering. This clustering shows that the dSSc, lSSc, morphea samples form distinct groups largely stratified by their clinical diagnosis. A. The unsupervised hierarchical clustering dendrogram shows the relationship among the samples using this list of 4,149 probes. Samples names have been color-coded by their clinical diagnosis: dSSc in red, lSSc in orange, morphea and EF in black, and healthy controls (Nor) in green. Forearm (FA) and Back (B) are indicated for each sample. Solid arrows indicate the 14 of 22 forearm-back pairs that cluster next to one another; dashed arrows indicate the additional 3 forearm-back pairs that cluster with only a single sample between them. Technical replicates are indicated by the labels (a), (b) or (c). 9 out of 14 technical replicates cluster immediately beside one another. B. Overview of the gene expression profiles for the 4,149 probes. Each probe has been centered on its median expression value across all samples analyzed. Measurements that are above the median are colored red and those below the median are colored green. The intensity of the color is directly proportional to the fold change. Groups of genes on the right hand side indicated with colored bars are shown in greater detail in panels C–H. C. Immunoglobulin genes expressed highly in a subset of patients with dSSc and in patients with morphea, D. proliferation signature, E. collagen and extracelluar matrix components, F. genes typically associated with the presence of T-lymphocyes and macrophages, G. Genes showing low expression in dSSc, H. Heterogeneous expression cluster that is high in lSSc and a subset of dSSc. In each case only a subset of the genes in each cluster are shown. The precise location of each gene in the cluster can be viewed in Supplemental Figure S1.
Mentions: We identified 4,149 probes whose expression varied from their median values in these samples by more than 2-fold in at least two of the 75 arrays and analyzed them by two-dimensional hierarchical clustering [18]. The resulting sample dendrogram shows that the samples separate into two main branches (Figure 1A) that in part stratify patients by their clinical diagnosis. The branch lengths in the tree are inversely proportional to the correlation between samples or groups of samples. The diversity in gene expression among the patients with scleroderma is greater than previously shown (Figure 1B) [16], [17] as distinct subsets of scleroderma are evident in the gene expression patterns. Some of these delineate existing classifications, such as the distinction between limited and diffuse, while others reflect new groups. One subset of dSSc patients cluster on the left branch (red) and has gene expression profiles that are distinct from both healthy controls and patients with lSSc (Figure 1B–1C), while a second subset of dSSc skin clusters in the middle of the dendrogram tree (black), and a third set clusters with healthy controls. We found lSSc samples formed a group in the middle portion of the dendrogram and could be associated with a distinct, but heterogeneous gene expression signature that also showed high expression in a subset of dSSc patients (Figure 1H). LSSc samples are partially intermixed with normal controls on the right boundary and with dSSc on the left boundary of the tree, illustrating that their gene expression phenotype is highly variable (Figure 1A). Samples taken from individuals with morphea also grouped together with a gene expression signatures that overlapped with those of dSSc and lSSc (Figure 1). Although nodes can be flipped, we have left the nodes of the dendrogram as originally organized by the clustering software, which places nodes with the most similar samples next to one another. Although, the assignment of samples into particular clusters (Table 3) would not change even if nodes were flipped.

Bottom Line: The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings.Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Dartmouth Medical School, Hanover, New Hampshire, United States of America.

ABSTRACT

Background: Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.

Methodology and findings: We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.

Conclusions and significance: Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

Show MeSH
Related in: MedlinePlus