Abstract / Summary
Background: Congenital cataract (CC) is an ophthalmic disorder diagnosed at birth or during early infancy, characterized by severe visual impairment and potential blindness. This study aimed to evaluate the pathogenicity of two variants: GJA8 c.201C>G (p.Asp67Glu) and CRYBB2 c.325A>T (p.Ile109Phe).
Methods: The patient's lenses were examined using slit-lamp photography and B-ultrasound. Whole-exome sequencing (WES) and sanger sequencing were performed to identify the variants. Multiple sequence alignment was conducted using CLUSTALW to assess the evolutionary conservation of the affected residues. Structural modeling and conformational analyzes of wild-type (WT) and mutant-type (MT) proteins were conducted using AlphaFold2, PyMOL, and computational structural dynamics approaches, including root mean square fluctuation (RMSF) and normal mode analysis (NMA).
Results: Two likely pathogenic missense variants were identified in two unrelated families: c.201C>G (p.Asp67Glu) in exon 2 of GJA8, which encodes Connexin 50 (Cx50) protein, was paternally inherited in family 1, whereas c.325A>T (p.Ile109Phe) in exon 4 of CRYBB2 occurred de novo in family 2. The c.201C>G (p.Asp67Glu) variant mapped to the first extracellular loop (EL1) of Cx50. Structural modeling revealed disruption of the Asp67-Arg185 ionic interaction (Cα-OD distance: 3.4 → 4.9 Å) alongside a novel hydrogen bond with Asn63. Reduced RMSF in the mutant indicated localized rigidification of EL1, potentially impairing channel gating and correlating with severe cataract in affected family members. The co-segregation of the variant with the affected phenotype within the family 1 was confirmed by sanger sequencing combined with phenotypic analysis. The c.325A>T variant (p.Ile109Phe) in CRYBB2 increased protein flexibility, and the bulky aromatic phenylalanine side chain perturbed the hydrophobic core of Greek key motif III, causing global dynamic instability. The CRYBB2 c.325A>T variant was further confirmed to be de novo by sanger sequencing in conjunction with phenotypic analysis in family 2. Furthermore, multiple sequence alignment demonstrated that both residues are highly evolutionarily conserved.
Conclusions: This study expands the pathogenic spectrum of congenital cataract. Our findings provide novel insights into the molecular diagnosis of CC and have important implications for genetic counseling.