A Novel CRYBB2 Splicing Mutation Is Associated With Lens Extracellular Matrix Remodeling and Vascular Alterations in Congenital Cataract

Purpose: We identify the genetic cause of autosomal dominant congenital cataract in a large family and define how a CRYBB2 splice-site variant perturbs lens development and associated ocular phenotypes. Methods: Whole-exome sequencing and Sanger sequencing were used for variant identification and segregation. Wild-type (WT) or mutant CRYBB2 was expressed in lens epithelial cells to assess splicing and downstream transcriptional changes by RNA sequencing and quantitative reverse transcription PCR (qRT-PCR). Extracellular matrix (ECM) organization and cell–matrix interactions were examined by immunostaining and adhesion assays. In zebrafish, crybb2 disruption was evaluated using lens structural assays, hyaloid vasculature imaging in Tg(flk1:EGFP) larvae, tracer distribution assays, and mRNA rescue. Single-cell RNA sequencing at 48 hours post-fertilization (hpf) was performed to profile lens and vascular populations and infer pathway-level changes. Results: We identified a novel CRYBB2 splice-site variant in affected members of a family with congenital cataract, consistent with an association with the disease in this pedigree. The variant disrupted normal splicing and was associated with altered βB2-crystallin–linked transcriptional programs and ECM homeostasis, including changes in collagen IV/laminin deposition and impaired cell–matrix adhesion. In zebrafish, crybb2 disruption caused lens fiber cell differentiation defects and was accompanied by abnormal hyaloid vascular patterning and altered tracer distribution around the lens; these phenotypes were partially improved by crybb2 mRNA rescue. Single-cell transcriptomic analyses suggested coordinated changes in ECM and junction/adhesion-related pathways across lens and vascular compartments, consistent with a lens-associated, non-cell-autonomous influence on adjacent vasculature. Conclusions: A novel CRYBB2 splice-site mutation is associated with autosomal dominant congenital cataract and disrupts lens homeostasis with accompanying changes in hyaloid vascular remodeling. These findings expand the pathogenic spectrum of CRYBB2 and support a model in which lens abnormalities are associated with altered lens–vascular interactions during development.