Background/Purpose: Limited cutaneous SSc (lcSSc) is characterised by less extensive skin fibrosis but patients can develop major internal organ complications and vascular manifestations. Gene expression analysis of SSc biopsies has been used to define molecular subsets of the disease and provide mechanistic insight into pathobiology.  Clinically uninvolved skin in the diffuse cutaneous subset has closely replicated gene expression signatures compared with biopsies from clinically involved skin and a number of vasoactive genes have been identified this way. We have investigated the gene expression abnormalities in the more prevalent lcSSc subset through detailed transcriptional analysis of skin biopsies taken from uninvolved forearm skin. 

Methods: Total RNA was extracted from skin biopsies from 15 patients with SSc fulfilling 2013 EULAR/ACR classification criteria with the limited subset of disease and 8 healthy controls (HC). Demographic, clinical and serological parameters were representative of the recruitment cohort but purposefully broad. Gene expression profiling was performed on a DNA oligonucleotide microarray chip.  Differentially expressed genes (DEG) were identified using Significance Analysis of Microarrays (SAM). Functional enrichment analysis of gene signatures was done via g:Profiler.

Results: There were 218 DEG between lcSSc and HC samples (False Discovery Rate<10%). 181/218 DEG were upregulated in lcSSc samples. Hierarchical clustering of DEG suggested the presence of 2 separate groups of lcSSc samples: ‘limited 1’ and ‘limited 2’. The ‘limited 1’ group (13 samples, 10 unique patients) showed upregulation of genes involved in cell adhesion, cardiovascular system (CVS) development (Figure 1) and extracellular matrix as well as immune and inflammatory response. The CVS development signature was of particular interest as its genes showed very strong enrichment in response to wounding, response to TGF-β and kinase cascade. Neither ‘limited 2’ group (6 samples, 5 unique patients) nor HC samples showed functional enrichment. There were no significant differences in demographic or clinical parameters between these two groups including the presence of significant microvascular involvement and hallmark antibody reactivities.

Conclusion: Our study suggests the presence of molecular subsets in lcSSc that overlap with other clinical and serological features based on gene expression profiling of biopsies from uninvolved skin. This may reflect important differences in pathogenesis within these patient groups. We identify differential expression of a subset of genes that relate to CVS and are enriched in fibrotic signalling.  This may shed light on underlying mechanisms of vascular disease in SSc. The enrichment in profibrotic profile is striking and suggests that dysregulated gene expression may contribute to vasculopathy and fibrosis in different disease subsets.