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Sarah J Garnai, David M Reed, Frank Joseph Giblin, Hemant S Pawar; Congenital cataract: functional effects of three CRYBB2 amino acid changes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1208. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to investigate the impact of three amino acid changes caused by a CRYBB2 gene conversion event that we identified in a large congenital cataract family.
The CRYBB2 cDNA was generated by RT-PCR using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), RNA from human retina, CRYBB2-specific primers and the pcDNA3.1 vector. Three mutations, c.433 C>T (p.R145W), c.440A>G (p.Q147R), and c.449C>T (p.T150M), were introduced into the wild type clone using the QuikChange Lightning kit (Agilent). Cultured human lens epithelium (HLE) SRA 01/04 cells were transfected using Lipofectamine 2000 (Life Technologies). Cell lysates containing protease inhibitors were sonicated and spun to generate separate supernatant and pellet fractions. After SDS-PAGE, Western blot analysis was carried out using a goat polyclonal antibody against CRYBB2 (Santa Cruz Biotechnology).
Missense changes R145W and T150 M were found to be conserved across 15 species while Q147R differs in puffer fish (Tetraodon nigroviridus). Protein modeling with PROVEAN and PolyPhen-2 predicted that R145W and T150M may each be deleterious/possibly damaging, while Q147R is predicted to be benign/neutral. ProtScale showed altered hydrophobicity for CRYBB2 containing all three missense changes. Cloned cDNA expression constructs were created containing wild type sequence and each of the three individual changes. We expressed wild type and triple mutant CRYBB2 cDNA in HLE cells. Western blot analysis showed that the wild type CRYBB2 protein is present only in the soluble fraction of the cell extract while the mutant protein is detected only in the pellet.
Informatic analysis suggests that two of the three CRYBB2 missense mutations that we have identified in a congenital cataract family are hitting residues that are conserved across species and predicted to be potentially deleterious. Initial studies using HLE cells indicate that although wild type CRYBB2 is soluble in the cytosol, the mutant protein carrying all three changes is aggregated and/or membrane-bound, leaving it in the cell pellet. Additional analysis of the effects of individual mutations will be presented.
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