Transcriptomic Evaluation of Juvenile Localized Scleroderma Skin With Histologic and Clinical Correlation

Abstract

Objective Juvenile localized scleroderma (LS) is an autoimmune disease of the skin whose pathogenesis is not well understood due to the rarity of the disease. This study was undertaken to determine the skin transcriptome in skin biopsy tissue from children with juvenile LS compared to pediatric healthy controls, with identification of significant molecular targets using RNA sequencing (RNA‐Seq). In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS, and were used to group the children into distinct genetic clusters based on immunophenotype. Methods RNA‐Seq was performed on sections of paraffin‐embedded skin tissue obtained from 28 children with juvenile LS and 10 pediatric healthy controls. RNA‐Seq was carried out using an Illumina HTS TruSeq RNA Access library prep kit, with data aligned using STAR and data analysis using a DESeq2 platform. A standardized histologic scoring system was used to score skin sections for the severity of inflammation and levels of collagen deposition. Histologic scoring was completed by 2 pathologists who were blinded with regard to the status of each sample. Spearman’s rank correlation coefficients were used to assess significant correlations between DEG expression profiles and skin histologic findings in patients with juvenile LS. Results We identified 589 significant DEGs in children with juvenile LS as compared to healthy controls. Hierarchical clustering was used to demonstrate 3 distinct juvenile LS immunophenotype clusters. The histologic scores of skin inflammation (based on numbers and categories of inflammatory cell infiltrates) were significantly correlated with the expression levels of HLA–DPB1, HLA–DQA2, HLA–DRA, and STAT1 genes (rs > 0.5, P < 0.01). Collagen thickness correlated with the expression levels of collagen organization genes as well as with genes found to be correlated with the severity of inflammation, including genes for major histocompatibility complex (MHC) class I, MHC class II, and interferon‐γ signaling. Conclusion Among children with juvenile LS, 3 distinct genetic signatures, or clusters, were identified. In one cluster, inflammation‐related pathways were up‐regulated, corresponding to the histologic skin inflammation score. In the second cluster, fibrosis‐related pathways were up‐regulated. In the third cluster, gene expression in the skin corresponded to the patterns seen in healthy controls. Up‐regulation of HLA class II genes was observed within the first cluster (characterized by predominant inflammation), a feature that has also been observed in the peripheral blood of patients with morphea and in the skin of patients with systemic sclerosis.

Morphea after initiation of dupilumab in two pediatric atopic dermatitis patients

“…In support of this hypothesis, flow cytometry studies show a Th1 inflammatory signature in localized scleroderma peripheral blood mononuclear cells 13 . Studies of transcripts of transcriptional profiles and cytokine levels suggest that levels of CXCL9/10/11 and IFN‐γ, which play a role in differentiation of Th1 cells, are higher in localized scleroderma patients with more inflammatory disease 14–17 . Increased macrophage infiltration, CXCL9, and IFN‐γ expression were seen in localized scleroderma tissue compared to healthy tissue and showed staining in close approximation with CXCR3 + T cells 15,17 .…”

“…13 Studies of transcripts of transcriptional profiles and cytokine levels suggest that levels of CXCL9/10/11 and IFN-γ, which play a role in differentiation of Th1 cells, are higher in localized scleroderma patients with more inflammatory disease. [14][15][16][17] Increased macrophage infiltration, CXCL9, and IFN-γ expression were seen in localized scleroderma tissue compared to healthy tissue and showed staining in close approximation with CXCR3 + T cells. 15,17 These studies may suggest that the imbalance in IFN-γ, CXCL9, and CXCL10 secreted by F I G U R E 1 Patients on dupilumab for atopic dermatitis.…”

Biomarkers in skin autoimmunity—An update on localised scleroderma

“…As it can already be seen in the work of Schutt et al (Figure 1c) with the downregulation of DNA methylation pathway in LS patients, 18 at the level of DNA genome, epigenetic regulation is another exciting field of study that also includes new findings for LS. Coit et al studied DNA methylation differences and similarities between juvenile systemic sclerosis and juvenile LS compared to matched healthy controls.…”

“…Next, Schutt et al analysed skin transcriptome in skin biopsy tissues from children with juvenile LS compared to paediatric healthy controls. 18 In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS and were used to group the children into distinct genetic clusters based on immunophenotype. RNA-Seq was performed on sections of paraffin-embedded skin tissue obtained from 28 children with juvenile LS and 10 paediatric healthy controls.…”

“…AR, androgen receptor; CSB, Cockayne syndrome complementation group B protein; GALNT12, N-acetylgalactosaminyltransferase 12; MHC, major histocompatibility complex; PD-1, programed death 1; PKN1, serine/ threonine protein kinase N1; PRC2, polycomb-repressive complex 2; rRNA, ribosomal RNA; SIRT1 sirtuin 1; TCA, tricarboxylic acid; TCR, T cell receptor; tRNA, transfer RNA. 18 signalling. Identifying 3 distinct genetic signatures and associated genes is a significant step forward in terms of diagnosing and differentiating LS from SSc.…”