Primary open angle glaucoma (POAG), the most prevalent subtype of glaucoma, is considered as a lifelong disease involving optic nerve deterioration with increased intraocular pressure (IOP) as a main risk factor. The specific pathogenesis of POAG has not been determined, but genetic background may contribute to its etiology. Genetic studies have identified several genes influencing POAG with MYOC being the first glaucoma-associated gene (Pang and Clark, 2020).

EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), also known as fibulin-3 (hereafter referred to collectively as fibulin-3), is an extracellular matrix (ECM) N-glycoprotein encoded by the EFEMP1 gene. EFEMP1 belongs to the Class Ⅱ fibulin (short fibulin) subfamily which is characterized by calcium-binding epidermal growth factor (cbEGF) domain tandem repeats and a C-terminal fibulin module (Livingstone et al., 2020; Yanagisawa et al., 2009). Fibulin-3 was shown to be widely present in adult tissues throughout the human body with abundant expression in ocular tissues (Livingstone et al., 2020). Several studies have reported associations of EFEMP1 variants with Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML) (Crowley et al., 2023; Garland et al., 2014) or age-related macular degeneration (Cheng et al., 2020). In addition, a missense variant c.418C > T, p.Arg140Trp in EFEMP1 has been identified in an African-American pedigree with autosomal dominant POAG and increased intracellular accumulation was found in HEK-293T cells overexpressing Trp140-mutant protein (Mackay et al., 2015). Gupta et al. have reported a rare association of EFEMP1 variants c.442C > T, p.Arg148Cys with autosomal dominant juvenile-onset open angle glaucoma in a patient with concomitant COL11A1-related Stickler syndrome (Gupta et al., 2023). Collantes et al. also reported EFEMP1 variants (c.238A > T, p.Asn80Tyr; c.1480T > C, p.Ter494Glnext*29; and c.1429C > T, p.Arg477Cys) which co-segregated in the pedigrees with juvenile open-angle glaucoma, and showed that these variants cause intracellular aggregation and retention of fibulin-3 protein in COS-7 cells (Collantes et al., 2022). More recently, an EFEMP1 variant c.175A > C p.Met59Leu was identified in two Chinese pedigrees diagnosed as POAG and this variant was found to induce intracellular protein aggregations, extracellular proteins secretion and changes to endoplasmic reticulum stress markers in HEK293T cells (Xiao et al., 2024). Interestingly, this study also found that intravitreal injection of pMet59Leu-expressing adenovirus 5 vector increased IOP in mice. Therefore, fibulin-3 may play an important role in physiology of ocular anterior segment tissues.

It is generally accepted that the actin cytoskeleton and its associated cellular interactions in the trabecular meshwork (TM) and juxtacanalicular tissues (JCT) mainly contribute to the formation of aqueous outflow resistance and IOP of the eye. The Rho GTPase (Rho)/Rho-associated kinase (ROCK) signaling pathway plays a crucial role in maintaining a bidirectional balance between actomyosin-regulated contractile activity and induced ECM synthesis thereby mediating cell and tissue stiffness within the conventional outflow pathway (Pattabiraman and Rao, 2010). As a secreted ECM protein, fibulin-3 was confirmed to be expressed in human TM and Schlemm's canal cells (Youngblood et al., 2021). Nevertheless, the specific function of fibulin-3 in TM cells is poorly understood, although previous studies (Stanton et al., 2017) have suggested that fibulin-3 interacts with enzymatic ECM regulators, such as tissue inhibitor of metalloproteinase-3 (TIMP-3; up-regulation) and matrix metalloprotease-9 (MMP-9; down-regulation), in posterior segment tissues controlling their expression. In non-ocular cells, fibulin-7 and fibulin-1 exhibit inhibitory effects on the RhoA protein, resulting in a disruption of stress fibers, decreased cell spreading, contraction of the cell body, and protrusion of dendritic extensions (de Vega et al., 2014; Williams and Schwarzbauer, 2009). These fibulin family proteins share a common fibulin-type module, indicating that fibulin-3 may exert similar functions in TM cell.

In this study, we report a stop codon variant (c.T1480C, p.X494Q) in EFEMP1 gene co-segregating with the phenotype of a Chinese POAG pedigree. Functional studies suggested that fibulin-3 encoded by EFEMP1 gene regulated the cytoskeleton assembly and ECM synthesis in trabecular meshwork cells via Rho/ROCK signaling, playing a role in the formation of normal or physiological resistance of aqueous humor outflow, while the EFEMP1 stop code variant disrupted the regulatory capacity of fibulin-3. Additionally, transforming growth factor-β1 (TGF-β1) can up-regulate fibulin-3 expression through activation of Rho/ROCK signaling. These results will contribute to understanding of the important role of fibulin-3 in the trabecular meshwork and may provide new therapeutic targets for POAG.