Overexpression of human γC-crystallin 5 bp duplication disrupts lens morphology in transgenic mice.

PURPOSE: To delineate the molecular mechanisms underlying autosomal dominant congenital cataracts caused by a 5 bp duplication in human CRYGC.
METHODS: c.119_123dup (CRYGC5bpd) and wild-type human γC-crystallin (CRYGC) were expressed in transgenic mouse lenses by the chicken βB1-crystallin promoter. Lenses were characterized histologically, by real-time PCR, SDS-PAGE, and Western blot. pET and Tet-on expression systems were used to express human CRYGC and CRYGC5bpd proteins in Escherichia coliand HeLa cells, respectively.
RESULTS: Transgenic expression of CRYGC5bpd mutant γC-crystallin results in nuclear cataracts in which lens fiber cells begin to show variable degrees of degeneration and vacuolization by postnatal day 21. By 6 weeks of age all CRYGC5bpd lenses exhibit abnormalities of varying severity, comprising large vacuoles in cortical fiber cells, swelling and disorganization of fiber cells, and defective fiber cell migration and elongation. Levels of CRYGC5bpd mRNA are 3.7- and 14.1-fold higher than endogenous Crygc mRNA in postnatal day 1 and 6-week CRYGC5bpd mice lens, respectively. Crygc, Crygb, Crybb2, and Crybb3 mRNA levels are decreased in CRYGC5bpd mice compared with wild-type and CRYGC mice. Both wild-type and mutant human γC crystallin are uniformly distributed in the cytosol of HeLa cells, but CRYGC5bpd is degraded when expressed in E. coli BL21(DE3).
CONCLUSIONS: Transgenic expression of mutant CRYGC5bpd γ-crystallin at near-physiological levels causes lens opacities and fiber cell defects, confirming the pathogenicity of this mutation. These results further suggest that HCG5pbd γ-crystallin causes cataracts through a direct toxic or developmental effect on lens cells causing damaged microstructure rather than through formation of HMW aggregates with resultant light scattering.