Two genes may serve as diagnostic biomarkers for keratoconjunctivitis sicca in patients with Sjögren’s syndrome.
In an integrated transcriptomic and experimental study, researchers used 2 publicly available gene expression datasets from the Gene Expression Omnibus. The GSE84844 dataset included whole-blood samples from 30 patients with Sjögren’s syndrome and 30 healthy controls, whereas GSE176510 contained conjunctival samples from 7 patients with keratoconjunctivitis sicca (KCS) and 19 healthy controls. The researchers screened 1,183 histamine-related genes using differential expression analysis and 2 machine learning algorithms, followed by laboratory experiments in immortalized human corneal epithelial cells.
The primary analyses sought to identify histamine-related genes associated with Sjögren’s syndrome–related KCS and evaluate their potential diagnostic value. Secondary analyses assessed immune cell infiltration, inflammatory signaling pathways, predicted transcriptional regulators, and gene-compound interactions. Laboratory experiments evaluated cell viability and inflammatory marker expression following targeted gene suppression under hyperosmotic stress.
Differential expression analysis identified 602 genes associated with Sjögren’s syndrome and 114 associated with KCS. After excluding genes with concordant expression changes in both datasets, the researchers identified 96 KCS-specific genes, including 39 associated with histamine-related pathways. Least absolute shrinkage and selection operator regression as well as support vector machine-recursive feature elimination identified IFITM1 and CTSS as the 2 genes selected by both algorithms.
Both genes demonstrated areas under the receiver operating characteristic curve (AUC) greater than 0.90 for distinguishing KCS samples from controls. A diagnostic nomogram combining the genes achieved an AUC of 1.00 in the training dataset, indicating perfect discrimination within the analyzed sample. However, the researchers emphasized that this result requires confirmation in larger, independent cohorts.
Laboratory experiments provided additional evidence of the genes’ involvement in ocular surface inflammation. The researchers used small interfering RNA to suppress IFITM1 and CTSS expression in immortalized human corneal epithelial cells, achieving knockdown efficiencies exceeding 70%. Under hyperosmotic stress, cells with either gene suppressed demonstrated statistically significantly better viability compared with stressed control cells. Suppression of either gene reduced expression of the inflammatory mediators interleukin-6 and tumor necrosis factor alpha under hyperosmotic conditions.
Further analyses linked the genes to distinct immune processes. IFITM1 was associated with antigen processing and presentation and major histocompatibility complex–related pathways, whereas CTSS was associated with positive regulation of type 2 immune responses. Analysis showed increased activated dendritic cells and T helper cells in KCS samples compared with controls. IFITM1 expression was positively correlated with activated dendritic cells, while CTSS expression was positively correlated with T helper cells, with correlation coefficients greater than 0.30.
Computational analyses also identified potential upstream regulatory mechanisms. STAT1 and STAT2 were predicted to regulate IFITM1, whereas SPI1 was identified as a potential regulator of CTSS. Molecular docking suggested potential interactions between CTSS and petesicatib and between IFITM1 and tetrachlorodibenzodioxin. These interactions did not establish therapeutic efficacy.
The findings were limited by the relatively small transcriptomic datasets, particularly the KCS cohort of 7 patients. Although the researchers used cross-validation and bootstrap resampling, the diagnostic model requires independent clinical validation. The analysis compared separate Sjögren’s syndrome and KCS datasets rather than longitudinally following patients, preventing conclusions about whether either gene predicts progression to ocular surface disease. The functional experiments were conducted in cultured corneal epithelial cells, and the proposed regulatory mechanisms require direct experimental confirmation. In vivo studies and multicenter clinical validation remain necessary.
“Further clinical and mechanistic validation is needed to establish their diagnostic robustness and to determine whether either gene represents a viable therapeutic target,” wrote lead study author NingLyu, of the Eye Institute and Department of Ophthalmology at the Eye & ENT Hospital atFudan University in China, and colleagues.
Full disclosures of the study authors can be found in the study.
Source: Eye Discovery
