Gene expression profiling of orbital adipose tissue in thyroid orbitopathy

Abstract

Purpose: Thyroid orbitopathy(TO) results from autoimmunity against orbital tissue. The underlying molecular mechanisms remain incompletely understood. We aim to determine differentially expressed genes that may be involved in stimulating orbital inflammation and fat expansion in TO using microarray gene expression profiling in a case control study.

Methods: Human orbital adipose samples were obtained during orbital decompression and upper lid surgeries in patients with TO. Patients with TO were subclassified as active(n=12) or inactive(n=21). Normal controls(n=21) were patients without autoimmune thyroid disease with adipose tissue harvested from corresponding anatomical locations at the time of unrelated orbital and lid surgeries.<br /> RNA was extracted then hybridized to Illumina humanHT12 v4 microarray. Expression signals were analyzed using R. Top differentially expressed genes were compared between active and inactive TO, and between active TO and normal controls ranked by fold change, level of expression and adjusted false discovery rate<0.05. Top ranked and some lower ranked genes of interest were validated by real time PCR in additional 8 active, 13 inactive TO and 11 normal controls. Gene set enrichment analysis(GSEA) was performed. Molecular pathways were analysed using DAVID bioinformatics.

Results: 721 probes representing 626 annotated genes were differerentially expressed in active compared to inactive TO. Defensins(DEFA1, DEFA1B, DEFA3) were overexpressed by 3.05-4.14 fold. TIMD4 was over-expressed by 4.3 fold. CD247, CD3A, CAMP, SLAM family member 6, GZMA, NKG7 were upregulated by 1.75-1.95 fold. Several markers for adipogenesis were overexpressed by 1.91-2.6 fold including SCD, FADS and ELOVL6. Increased expression of FADS1 and SCD were confirmed in active TO versus normal controls.<br /> GSEA revealed a significant proportion of genes upregulated in gene sets tyrosine kinase protein CSK pathway, cytotoxic T lymphocyte, T cell apoptosis, glycolysis, IL-12, T helper, T cell receptor activation, caspase cascade, TOB1, cell to cell adhesion signaling and strathmin pathways. Molecular pathway analysis correlated well with GSEA analysis.

Conclusions: Active TO is marked by up-regulation of genes involved in cellular, innate immune and inflammatory response and enhanced orbital adipogenesis.TIMD4, DEFA1, DEFA1B and DEFA3 may be involved in stimulating immune mediated orbital inflammation.