Abstract
High-throughput RNA sequencing (RNA-seq) uses massive parallel sequencing technology, allowing the unbiased analysis of genome-wide transcription levels and tumor mutation status. Immunoglobulin G4-related ophthalmic disease (IgG4-ROD) is a fibroinflammatory disease characterized by the enlargement of the ocular adnexal tissues. We analyzed RNA expression levels via RNA-seq in the biopsy specimens of three patients diagnosed with IgG4-ROD. Mucosa-associated lymphoid tissue (MALT) lymphoma, reactive lymphoid hyperplasia (RLH), normal lacrimal gland tissue, and adjacent adipose tissue were used as the controls (n = 3 each). RNA-seq was performed using the NextSeq 500 system, and genes with |fold change| ≥ 2 and p < 0.05 relative to the controls were defined as differentially expressed genes (DEGs) in IgG4-ROD. To validate the results of RNA-seq, real-time polymerase chain reaction (PCR) was performed in 30 IgG4-ROD and 30 orbital MALT lymphoma tissue samples. RNA-seq identified 35 up-regulated genes, including matrix metallopeptidase 12 (MMP12) and secreted phosphoprotein 1 (SPP1), in IgG4-ROD tissues when compared to all the controls. Many pathways related to the immune system were included when compared to all the controls. Expressions of MMP12 and SPP1 in IgG4-ROD tissues were confirmed by real-time PCR and immunohistochemistry. In conclusion, we identified novel DEGs, including those associated with extracellular matrix degradation, fibrosis, and inflammation, in IgG4-ROD biopsy specimens. These data provide new insights into molecular pathogenetic mechanisms and may contribute to the development of new biomarkers for diagnosis and molecular targeted drugs.
Highlights
Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a relatively new disease concept that was originally proposed in Japan
We investigated the expression of RNA in lesions of IgG4-ROD in biopsy specimens compared with various controls comprising adipose tissues adjacent to the IgG4-ROD lesion, orbital mucosa-associated lymphoid tissue (MALT) lymphoma, reactive lymphoid hyperplasia (RLH), and normal lacrimal gland tissues to search for new biomarkers and elucidate the pathophysiology of IgG4-ROD
These genes were segregated clearly into low and high expression groups for IgG4-ROD specimens but showed widely variable distributions with no consistent patterns in all the control tissues, indicating that the cluster analysis using differentially expressed genes (DEGs) separated IgG4-ROD from other diseases and normal tissues. These results indicate a possibility that these genes may contain diagnostic and/or therapeutic biomarkers or important factors contributing to the pathological condition of IgG4-ROD
Summary
Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a relatively new disease concept that was originally proposed in Japan. Involvement of follicular T cells (Tfh) and regulatory T cells (Tregs) has been reported [2,3], but the pathogenetic mechanisms remain to be elucidated. Various etiologies, such as infectious diseases [4], have been proposed. Diagnostic imaging, histology, molecular analysis, and flow cytometry using biopsy specimens are the most commonly used diagnostic tools for orbital lymphoproliferative disorders [5,6]. Several reports have suggested that 12% of orbital MALT lymphoma result from a background of IgG4-ROD [11,12]
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