Identification of differentially expressed genes in fibroblasts derived from patients with Dupuytren's Contracture

Abstract

Dupuytren's contracture (DC) is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytren's patients. Total RNA isolated from diseased palmar fascia (DC) and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group) was subjected to quantitative analyses using two different microarray platforms (GE Code Linktrade mark and Illuminatrade mark) to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM) software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLinktrade mark and Illuminatrade mark platforms, respectively. The CodeLinktrade mark platform identified 18 upregulated and 51 downregulated genes. Using the Illuminatrade mark platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4), fibulin-1 (FBLN-1) transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytren's contracture and may ultimately serve as attractive molecular targets for alternative therapies.

Figure 1

The relative decrease in gene expression levels for PRG4, FBLN-1 and Type XV collagen in DC derived fibroblasts as determined by real-time RT-PCR. Values are mean ± SEM of three independent studies, each performed in duplicates. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ C_t values between carpal tunnel and DC derived fibroblasts.

Figure 2

The relative decrease in gene expression levels for PRG4, FBLN-1 and Type XV collagen in DC derived fibroblasts as determined by real-time RT-PCR. Values are mean ± SEM of three independent studies, each performed in duplicates. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ C_t values between carpal tunnel and DC derived fibroblasts.

Figure 3

The relative decrease in gene expression levels for PRG4, FBLN-1 and Type XV collagen in DC derived fibroblasts as determined by real-time RT-PCR. Values are mean ± SEM of three independent studies, each performed in duplicates. GAPDH was used as an internal control. Statistical analyses were performed by Student's t test. Relative quantification of gene expression was calculated by comparing δ C_t values between carpal tunnel and DC derived fibroblasts.