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.