RNA Sequencing Identifies Novel, Specific Biomarkers in Pterygia

The underlying causes for the development of pterygia are not entirely understood, although there are confirmed environmental and demographic risk factors associated with the disease. A recent study published in Frontiers in Medicine used MACE RNA sequencing to provide more insight into the pathogenesis and treatment of pterygia.

The disease is characterized by growth of epithelial and fibrovascular conjunctival tissue on the cornea in a wing shape, potentially causing vision impairment resulting from induced astigmatism and optical axis involvement. It is common, with a worldwide prevalence of approximately 12%, and older males are at a higher risk of developing pterygia. Ultraviolet light exposure is also a risk factor.

Pterygia treatment currently involves surgical removal and autologous conjunctival transplantation in combination with cytostatic and/or immunomodulatory therapy. But while adjuvant therapy has reduced recurrence rates, approximately 5% of patients experience recurrence post-treatment.

Epithelial mesenchymal transition, immunological and anti-apoptotic mechanisms, viral infections, angiogenic stimulation, and dysregulation of growth factors are all potential mechanisms of pterygia that have been discussed. However, the underlying causes aren’t entirely clear.

“To date, a number of studies have used microarray technology to analyze the expression profile of pterygium samples, a method which is limited by technical issues, including limited probe coverage, inconsistent probe hybridization efficiency and its insensitivity to transcripts of low abundance,” the authors wrote. RNA sequencing, on the other hand, is a more accurate and unbiased way to analyze gene expression and can detect novel and rare transcripts that may previously have gone undetected.

The retrospective study analyzed the cellular microenvironment and transcriptional profile using a total of 26 conjunctival samples, including 8 pterygium and 8 healthy conjunctival specimens for transcriptome analysis and 7 pterygium and 3 healthy control samples for immunohistochemistry.

To identify pterygium-specific markers, recently published transcriptional profiles from 26 patients with neoplastic conjunctival lesions, including 12 with conjunctival melanoma, 7 with squamous cell carcinoma (SCC), and 7 with papilloma were included to identify pterygium-specific markers.

Unsupervised transcriptome analysis showed distinct differences between pterygium samples versus healthy conjunctiva. Cell type enrichment analysis showed that in pterygia, the cellular microenvironment was mainly characterized by smooth muscle cell enrichment. Type 2 T-helper cells (Th2), CD4+ memory T-cells, classical dendritic cells, CD8+ T-cells, and M2 macrophages also showed enrichment in pterygia cells.

Given the predominance of smooth muscle cell enrichment, known markers of smooth muscle cells and of myofibroblasts were assessed. Myofibroblasts were the most differentially enriched cell type in pterygium tissue, which suggests they have a role in the pathogenesis of pterygia. Study authors note, “Current evidence suggests that myofibroblasts emerge from conjunctival epithelial cells through the process of epithelial-mesenchymal transition.”

T-cells were the most predominantly enriched immune cells in samples of pterygia tissue. Previous studies have found the same, which both validates the transcriptome analysis conducted for this study and suggests potential therapeutic targets.

SPARC was one of the most specific pterygia marker genes. Upon protein expression analysis of SPARC in pterygia and control cells, researchers found that there was distinct epithelial and stromal immunoreactivity against SPARC as well as more prominent staining in the vessels of pterygia tissue versus controls. Recent research has shown silencing of SPARC inhibits the expression of markers known to be profibrotic, suggesting SPARC may be another potential target for pterygia treatment.

In all, the study identified 450 marker genes that were also specific for pterygia in the validation data. Although limited by its retrospective single-center design and use of bulk RNA sequencing rather than single-cell RNA sequencing, which is more accurate but was not possible with the formalin-fixed and paraffin-embedded samples, the study authors conclude that the findings validate key cellular microenvironment characteristics associated with pterygia.

“The results of this study contribute to an improved understanding of the pathophysiological processes underlying the disease and reveal new diagnostic biomarkers that may enable new options of targeted therapy for pterygia,” the authors concluded.

Reference

Wolf J, Hajdu RI, Boneva S, et al. Characterization of the cellular microenvironment and novel specific biomarkers in pterygia using RNA sequencing. Front Med (Lausanne). Published online January 31, 2022. doi:10.3389/fmed.2021.714458