Molecular Pathogenesis of Papillo-Renal Syndrome

Abstract

Purpose:: Papillo-renal syndrome (PRS) is an autosomal dominant disorder characterized by congenital anomalies of the optic nerve and kidney. Mutations in the developmentally-regulated transcription factor gene PAX2 have been described in many cases. Although most of these mutations are predicted to result in functional haploinsufficiency (e.g., by nonsense-mediated decay), a cluster of missense mutations in the paired domain sequence of PAX2 have been reported, suggesting that this area of the protein may have special functional significance. We therefore investigated the functional effect of these mutations in in vitro systems.

Methods:: Using site-directed mutagenesis, three previously-reported human mutations (Mutant #1 cG223A, pG75C, Mutant #2 c222insGAGACC, p74ins GluThr, Mutant #3 cG210C, pR71T and cG223A, pG75C) were introduced in a Pax2 expression construct driven by the CMV promoter. In addition, a missense mutation in the same region of Pax2 found in a mouse model of PRS (gA220G, pT74A) was investigated. The ability of these mutant Pax2 proteins to trans-activate a luciferase reporter gene under the control of a Pax2-inducible element was compared to that with wild-type protein in NIH 3T3 cells. Levels of protein expression were compared using Western blot analysis and sub-cellular localization was evaluated using immunofluorescence.

Results:: Internal control corrected luciferase activity was reduced in all four mutants compared to the wild type. The T74A change results in approximately 50% reduction of Pax2 expression and a commensurate reduction in transactivation. Although wild-type protein was exclusively localized to the nucleus, mutant protein could be identified in the cytoplasm as well as the nucleus. Similar studies are currently underway to evaluate the other mutants.

Conclusions:: The reported paired domain missense mutations in Pax2 appear to result in hypomorphic alleles. The clustering of mutations in this region of the protein suggests it has important physiologic function. Further, we are investigating the potential protein-protein interactions in this region.

Keywords: transcription factors • optic nerve • pathobiology