Mechanism for Dominant and Recessive Cataracts Caused by Point Mutations of AlphaA–crystallin Gene

Abstract

Abstract: : Purpose: The goal of our study is to elucidate the molecular basis for how and why different point mutations of aA–crystallin gene lead to distinct dominant and recessive cataract phenotypes. Methods: Mouse mutations were determined by a genome wide linkage analysis. RT–PCR products of both mutant lens mRNAs were sequenced to define the mutations in the aA–crystallin gene. The properties of mutant lenses were examined by histology and biochemical methods. Results: Mouse L1N line, an ethylnitrosourea (ENU)–induced mutation, develops dominant cataract; while mouse nm3365 line, a spontaneously occurred mutation, shows recessive inheritance. Both mutations are mapped to chromosome 17 near the vicinity of aA–crystallin gene and subsequently verified to be missense mutations of aA–crystallin – Tyr118Asp mutation for L1N and Arg54Cys mutation for nm3365. Histopathology shows abnormal epithelial cells and insufficiently elongated fibers in the neonatal nm3365 lenses, and intracellular vacuoles in the lens fibers of nm3365 embryonic lenses at 16.5 dpc. In contrast, both heterozygous and homozygous L1N lenses have relative normal epithelium and fibers. However, gel–filtration analysis reveals the disappearance of a–crystallin peak and an obvious increase of high molecular weight peak in the water–soluble proteins of L1N homozygous lenses. Conclusions: The aA–crystallin Tyr118Asp and Arg54Cys mutations lead to distinct cataract phenotypes and each mutation has its own unique morphological or biochemical changes. It is a surprise that Arg54Cys mutation in the nm3365 mouse line occurs at the same residue of a previously described Arg54His mutation (Lop18), both mutants cause recessive cataract. It is unlikely that Arg54Cys or Arg54His mutation affect the stability of aA–crystallin since no cataract is observed in their heterozygous mice that express mutated subunits, although both homozygous mutant mice develop severe cataracts. Thus, we hypothesize that the Arg54 residue must be essential for substrate selectivity and/or chaperone function of aA–crystallin but dispensable for its protein stability, while the Tyr118 residue is important for the stability of aA–crystallin in the lens. The dominant cataract of mouse L1N line is likely due to abnormal protein aggregations that are caused by the expression of unstable aA–crystallin–Y118D subunits in the lens.

Keywords: crystallins • chaperones • cataract