A Variant in the 5'UTR of the Cone Gene Encoding the Cone Gamma Subunit of cGMP-PDE that Causes Overexpression of the Protein; Disease-causing Mutation or Rare Polymorphism?

Abstract

Abstract: : Purpose: During exon-screening of 238 patients with various forms of cone-related retinal degeneration, a G to C substitution in the 5'UTR of the gene encoding the gamma subunit of cone cGMP-PDE (PDEγ) was found in the DNAs of two siblings with cone-rod dystrophy, and was not found in 92 control DNAs. Our purpose was to determine if this substitution could be responsible for disease by investigating its effect on protein synthesis. Methods: Two expression constructs were made by subcloning 5'UTR DNAs from normal or variant PDEγ into the pGEM3Z vector in front of the luciferase coding region. Equal amounts of in vitro synthesized transcripts were used for in vitro translation. The amount of protein synthesized was quantified by measuring luciferase activity. mRNA sequences were analyzed for the presence of stem-loop structures using the "RNA Secondary Structure Prediction Program" of Genebee Sevices, which determines the most stable structures and their free energy levels. Results: The luciferase activity (the efficiency of protein synthesis) of the construct containing the variant 5'UTR was 5-6-fold higher than that of the wild type construct. The predicted secondary structure of the cone PDEγ 5'UTR indicated that the region containing the G to C substitution forms a stem-loop structure that may be involved in RNA-protein interactions, and therefore, may play an important role in the regulation of protein synthesis. Conclusions: Our results suggest that the G to C substitution leads to upregulation of cone PDEγ synthesis in the photoreceptor cells. Our hypothesis is that the excess of PDEγ inhibits the activation of the cone cGMP-PDE enzyme leading to pathogenic levels of cGMP in cones, similar to what happens in the rods of rd1 mouse due to a mutation in the catalytic beta subunit of rod cGMP-PDE.

Keywords: gene/expression • candidate gene analysis • molecular biology