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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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Quantitative Real Time PCR analysis of representative biopsies.The mRNA levels of three genes, TNFRSF12A (A), CD8A (B) and WIF1 (C) were analyzed by Taqman quantitative real time PCR. Each was analyzed in two representative forearm skin biopsies from each of the major subsets of proliferation, inflammatory, limited and normal controls. In the case of TNFRSF12A, patient dSSc11 was replaced by patient dSSc10, which cluster next to one another in the intrinsic subsets and show similar clinical characteristics (Table 1). Each qRT-PCR assay was performed in triplicate for each sample. The level of each gene was then normalized against triplicate measurements of GAPDH to control for total mRNA levels (see materials and methods). The relative expression values are displayed as the fold change for each gene relative to the median value of the eight samples analyzed.
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pone-0002696-g007: Quantitative Real Time PCR analysis of representative biopsies.The mRNA levels of three genes, TNFRSF12A (A), CD8A (B) and WIF1 (C) were analyzed by Taqman quantitative real time PCR. Each was analyzed in two representative forearm skin biopsies from each of the major subsets of proliferation, inflammatory, limited and normal controls. In the case of TNFRSF12A, patient dSSc11 was replaced by patient dSSc10, which cluster next to one another in the intrinsic subsets and show similar clinical characteristics (Table 1). Each qRT-PCR assay was performed in triplicate for each sample. The level of each gene was then normalized against triplicate measurements of GAPDH to control for total mRNA levels (see materials and methods). The relative expression values are displayed as the fold change for each gene relative to the median value of the eight samples analyzed.

Mentions: In order to validate the gene expression in the major groups found in this study, we performed quantitative real time PCR (qRT-PCR) on three genes selected from the intrinsic subsets (Figure 7). These included TNFRSF12A, which is highly expressed in the dSSc patients and shows high expression in patients with increased MRSS (see Figure 6), WIF1, which shows low expression in SSc its decreased expression is associated with increased MRSS, and CD8A, which is highly expressed in CD8+ T cells and is highly expressed in the inflammatory subset of patients. A representative sampling of patients from the intrinsic subsets was analyzed for expression of these three genes. Each was analyzed in triplicate and standardized to the expression of GAPDH.


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)

Quantitative Real Time PCR analysis of representative biopsies.The mRNA levels of three genes, TNFRSF12A (A), CD8A (B) and WIF1 (C) were analyzed by Taqman quantitative real time PCR. Each was analyzed in two representative forearm skin biopsies from each of the major subsets of proliferation, inflammatory, limited and normal controls. In the case of TNFRSF12A, patient dSSc11 was replaced by patient dSSc10, which cluster next to one another in the intrinsic subsets and show similar clinical characteristics (Table 1). Each qRT-PCR assay was performed in triplicate for each sample. The level of each gene was then normalized against triplicate measurements of GAPDH to control for total mRNA levels (see materials and methods). The relative expression values are displayed as the fold change for each gene relative to the median value of the eight samples analyzed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481301&req=5

pone-0002696-g007: Quantitative Real Time PCR analysis of representative biopsies.The mRNA levels of three genes, TNFRSF12A (A), CD8A (B) and WIF1 (C) were analyzed by Taqman quantitative real time PCR. Each was analyzed in two representative forearm skin biopsies from each of the major subsets of proliferation, inflammatory, limited and normal controls. In the case of TNFRSF12A, patient dSSc11 was replaced by patient dSSc10, which cluster next to one another in the intrinsic subsets and show similar clinical characteristics (Table 1). Each qRT-PCR assay was performed in triplicate for each sample. The level of each gene was then normalized against triplicate measurements of GAPDH to control for total mRNA levels (see materials and methods). The relative expression values are displayed as the fold change for each gene relative to the median value of the eight samples analyzed.
Mentions: In order to validate the gene expression in the major groups found in this study, we performed quantitative real time PCR (qRT-PCR) on three genes selected from the intrinsic subsets (Figure 7). These included TNFRSF12A, which is highly expressed in the dSSc patients and shows high expression in patients with increased MRSS (see Figure 6), WIF1, which shows low expression in SSc its decreased expression is associated with increased MRSS, and CD8A, which is highly expressed in CD8+ T cells and is highly expressed in the inflammatory subset of patients. A representative sampling of patients from the intrinsic subsets was analyzed for expression of these three genes. Each was analyzed in triplicate and standardized to the expression of GAPDH.

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