Abstract
Purpose: :
A 5bp duplication in CRYGC (c.123_128insGCGGC) has previously been associated with autosomal dominant congenital cataracts in humans. Here we confirm the causal nature of this association and explore the mechanism by which this mutation might cause cataracts as well as begin to elucidate the role of γC–Crystallin in the lens.
Methods: :
The mutant human γC–crystallin with a 5–bp duplication (HGC5bpd) and a normal human γC–crystallin (HGC) serving as a control were expressed in transgenic mice driven by chicken ßB1–Crystallin promoter. The morphology and histology of mice transgenic for the mutant and normal γC–crystallin were characterized. Expression of the transgene was analyzed by Real–Time PCR, SDS–PAGE and Western blot analysis. A model of the HGC5bpd mutant protein was predicted based on homology to α–cobratoxin.
Results: :
In mice transgenic for HGC5bpd (OG02) the lens initially develops normally, but the fiber cells begin to show variable degrees of degeneration and vacuolization by 21 days of life and large lacunae and general degeneration of the lens is apparent by 6 weeks of age. Western blot analysis of lens extracts confirms that the mutant protein is present in transgenic mouse lenses. Real–time quantitative PCR shows that levels of the HGC5bpd mRNA are 3.7–fold and 14.1–fold higher than endogenous γC–Crystallin mRNA in postnatal day 1 and 6–week old OG02 mice lenses respectively, but are similar to those of transgenic HGC mRNA in OGO4 mice. mRNA levels of γC–, γB–, ßB2– and ßB3–crystallins in 6–week OG02 mice appear to be decreased compared to those of wild type (WT) mice. However, the levels in lenses of 4–week old OG04 mice are similar to those in WT. Sequence alignment of the HGC5bpd mRNA with α–cobratoxin shows 50% similarity, and three–dimensional protein prediction suggests that they might have similar tertiary structures.
Conclusions: :
Expression of HGC5bpd causes cataracts in transgenic mice, confirming the causative nature of this mutation with cataracts in humans. Lens histology suggests that the mechanism of cataractogenesis involves a toxic effect on the lens fiber cells with consequent disruption of normal lens morphology, and homology of the mutant HGC5bpd with α–cobratoxin provides a rationale for this observation.
Keywords: transgenics/knock-outs • cataract