We discovered disease-associated variants in the gene neurobeachin-like 2 (NBEAL2) in two families with keratoconus (KC), a degenerative, thinning disease of the cornea. NBEAL2 encodes a cellular scaffold protein (2,750 amino acids, 302 kDa) whose function in the cornea is unknown. We propose to elucidate the role of NBEAL2 in the cornea. The protein is present in platelets, and NBEAL2 variants, other than the ones we identified in KC, cause an extremely rare hematological disease known as gray platelet syndrome (GPS). Patients with GPS have low platelets that are defective in granular protein secretion. Nbeal2-/- mice, which systemically lack Nbeal2, also show platelet anomalies, but their eyes have not been examined. The NBEAL2 protein has a concanavalin-A lectin-like domain near the N-terminus, within which we detected an arginine-to-glutamate substitution (R659Q) in one family with KC. The second family with KC carries a valine- to-isoleucine substitution (V2118I) in a highly conserved domain of NBEAL2 called the Beige and Chediak- Higashi (BEACH) domain, found in only eight other proteins. BEACH proteins regulate vesicular secretion, lysosomal functions, membrane dynamics, and signaling. Mutations in BEACH proteins have been associated with hematologic diseases, hypopigmentation, exfoliation syndrome, neurodegeneration, wound healing, and cancer, but are understudied in eye diseases. A single recent study reported that NBEAL2 is tethered to vesicle membranes in platelets where it interacts with DOCK7, SEC16A, and other proteins to modulate actin polymerization, protein transport, and secretion. Our preliminary data show the presence of NBEAL2 in the epithelium and the corneal stroma, raising the question: what is the role of NBEAL2 in the cornea, such that its variants contribute to KC? A major function of cornea-resident cells is the maintenance of a specialized barrier tissue through effective secretion of basement membrane and stromal extracellular matrix (ECM) proteins and collagens. We hypothesize that, in resident corneal cells, NBEAL2 regulates secretion of ECM proteins and collagens necessary for corneal integrity. We will test our central hypothesis in two aims.
Aim I will examine corneas of Nbeal2-/- mice under homeostatic conditions and after epithelial debridement injuries. The wound healing response is orchestrated by epithelial and stromal cells that repair the basement membrane and the stroma through ECM secretion, and this secretion mechanism may require NBEAL2.
Aim II will test NBEAL2-mediated secretory functions in human corneal epithelial and stromal cell cultures after perturbation of endogenous NBEAL2 or expression of NBEAL2 KC variants. Our findings will elucidate functions of NBEAl2, a BEACH protein, novel to the cornea. Mechanistic insights from this study will identify targets that might be manipulated for increased ECM production and corneal integrity in KC.

Public Health Relevance

Keratoconus (KC) is a genetically complex, site threatening disease that affects 1 in 2000, primarily young people causing extensive health and economic burden. We identified KC-associated DNA variants in NBEAL2, novel to the eye. We will test corneal functions of NBEAL2 using donor human corneal cell cultures and Nbeal2-/- mice for future therapeutic KC targets.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1)
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Mckie, George Ann
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New York University
Schools of Medicine
New York
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