Single-cell transcriptomics in thyroid eye disease

Abstract

Thyroid eye disease (TED) is a poorly understood autoimmune condition affecting the retroorbital tissue. Tissue inflammation, expansion, and fibrosis can potentially lead to debilitating sequelae such as vision loss, painful eye movement, proptosis, and eyelid retraction. Current treatment modalities for TED include systemic glucocorticoids, thioamides, methimazole, teprotumumab, beta-blockers, and radioactive iodine; however, it has been reported that up to 10%-20% of TED patients relapse after treatment withdrawal and 20%-30% are unresponsive to mainstay therapy for reasons that have yet to be more clearly elucidated. In the past 4 years, vision researchers have harnessed high-throughput single-cell RNA sequencing to elucidate the diversity of cell types and molecular mechanisms driving the pathogenesis of TED at single-cell resolution. Such studies have provided unprecedented insight regarding novel biomarkers and therapeutic targets in TED. This timely review summarizes recent breakthroughs and emerging opportunities for using single-cell and single-nuclei transcriptomic data to characterize this highly complex disease state. We also provide an overview of current challenges and future applications of this technology to potentially improve patient quality of life and facilitate reversal of disease endpoints.

Keywords: Gene expression; Graves’; orbitopathy; single-cell RNA sequencing; single-nuclei RNA sequencing; thyroid eye disease; thyroid-associated ophthalmopathy; transcriptome.

Figure 1

Schematic representation of single-cell RNA sequencing (scRNA-seq) experimental design and applications in thyroid eye disease (TED). (a) Orbital connective tissue samples from TED patients are obtained during orbital decompression surgery. Cells undergo enzymatic lysis and dissociation into individual micro-environments to release mRNA. (b) Downstream processing of mRNA includes conversion to complementary DNA (cDNA) vis-à-vis reverse transcription, cDNA amplification either by the polymerase chain reaction (PCR) or in vitro transcription, cDNA library preparation and sequencing, and quality control (not shown) to assess technical variability and cell viability. (c) Computational tools are then used to assess transcriptional similarity of cells, which are grouped into distinct clusters based on differential gene expression of canonical lineage markers. In two landmark TED scRNA-seq studies, orbital fibroblasts and CD4⁺ T cell subtypes have been extensively characterized. (d) Applications of scRNA-seq in TED are wide-ranging and consist of characterizing the orbital micro-environment, uncovering cell-cell interactions, signaling pathways, and pseudotime analysis. Created with BioRender.com