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Gene Expression Profiling of Scleroderma Skin

ABSTRACT: We have analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from 34 subjects: 17 patients with SSc with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea and 6 healthy controls. In total, 61 skin biopsies were analyzed. The addition of 14 technical replicates resulted in a total of 75 microarray hybridizations. The Intrinsic_scleroderma_genes.txt supplementary file lists the 995 genes with the most consistent expression between each forearm-back pair and technical replicates, but with the highest variance across all samples analyzed. An intrinsic gene identifier algorithm was used to select a set of intrinsic scleroderma genes. A total of 34 experimental groups were defined, each representing the 34 different subjects in our study. Replicate hybridizations for a given patient were assigned to the same experimental group. Each gene was analyzed and assigned a score that is inversely related to how intrinsic the gene's expression is across the samples analyzed. The analysis was repeated on randomized data in order to estimate a False Discovery Rate (FDR). An intrinsic score of 0.3 selected 995 genes with an FDR of 4% (39 ± 7 genes) while retaining reproducible clustering of technical replicates. Keywords: global gene expression profiling using Agilent Whole Human genome oligo arrays

ORGANISM(S): Homo sapiens

PROVIDER: GSE9285 | GEO | 2008/07/15

SECONDARY ACCESSION(S): PRJNA102919

REPOSITORIES: GEO

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Gene Expression Profiling of Scleroderma Skin

Project description:We have analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from 34 subjects: 17 patients with SSc with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea and 6 healthy controls. In total, 61 skin biopsies were analyzed. The addition of 14 technical replicates resulted in a total of 75 microarray hybridizations. The Intrinsic_scleroderma_genes.txt supplementary file lists the 995 genes with the most consistent expression between each forearm-back pair and technical replicates, but with the highest variance across all samples analyzed. An intrinsic gene identifier algorithm was used to select a set of intrinsic scleroderma genes. A total of 34 experimental groups were defined, each representing the 34 different subjects in our study. Replicate hybridizations for a given patient were assigned to the same experimental group. Each gene was analyzed and assigned a score that is inversely related to how intrinsic the gene's expression is across the samples analyzed. The analysis was repeated on randomized data in order to estimate a False Discovery Rate (FDR). An intrinsic score of 0.3 selected 995 genes with an FDR of 4% (39 Â± 7 genes) while retaining reproducible clustering of technical replicates. Keywords: global gene expression profiling using Agilent Whole Human genome oligo arrays common reference design; we used Universal Human Reference RNA (Stratagene) as our reference for every array; For every hybridization sample RNA was always labeled with Cy3, and reference RNA was always labeled with Cy5; Microarray hybridizations were performed for 61 RNA samples from 34 subjects resulting 61 hybridizations. Fourteen replicate hybridizations were added, resulting in a total of 75 microarray hybridizations.

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Project description:Scleroderma is an autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy and fibrosis. We have previously demonstrated that low Fli1 expression in scleroderma fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc, playing key roles in disease pathogenesis However, a role for immune Fli1 in SSc has not yet been described. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in scleroderma. Comparison of the expression of Fli1 in monocytes, B- and T-cell fractions of PBMCs isolated from scleroderma patients and healthy controls, showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from Fli1flox/floxLysMCre+/+ mice, and found that markers of alternative macrophage activation were increased with Fli1 deletion. Coculture of Fli1-deficient myeloid cells and primary human or mouse fibroblasts resulted in a potent induction of collagen type I, independent of TGFβ upregulation. We next analyzed global gene expression profile in response to Fli1 downregulation, to gain further insight into the molecular mechanisms of this process and to identify differentially expressed genes in myeloid cells. Of relevance to SSc, the top most upregulated pathways were hallmark IFN-ɣ and IFN-α response. Additionally, several genes previously linked to SSc pathogenesis and fibrosis in general were also induced, including MCP-1, MCP-3, MMP12, and CXCL10. ANKRD1, a profibrotic transcription co-regulator was the top upregulated gene in our array. Our results show that Fli1-deficient myeloid cells share key features with cells from SSc patients, with higher expression of profibrotic markers and activation of interferon responsive genes, thus suggesting that low Fli1 may contribute to SSc pathogenesis.

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