This competing renewal application for the grant, Model Systems for PXE (AR R0155225) revolves around pseudoxanthoma elasticum (PXE), the paradigm of heritable aberrant mineralization disorders, manifesting in the skin, eyes, and the cardiovascular system with considerable morbidity and mortality. PXE is caused by mutations in the ABCC6 gene, encoding an efflux transporter expressed primarily in the liver. Our recent data, based primarily on experimentation on Abcc6-/- mice, which serve as a model for PXE, have suggested that PXE is a metabolic disorder at the genome/environment interface. One of the clinical features of PXE is considerable intra- and interfamilial heterogeneity. Our previous attempts to establish direct genotype/phenotype correlations in a cohort of ~300 patients have been unsuccessful, suggesting phenotypic modulation by genetic and environmental factors. This application has two specific areas of investigation. The first one focuses on identification of phenotypic modifier genes by mouse genomics through quantitative trait loci analysis. These analyses take advantage of our recent discovery of four inbred mouse strains that harbor the same ABCC6 mutation in their genome, yet the phenotypic expression is highly variable. These mouse strains, in the context of Abcc6-/- mice and their wild- type counterparts, will be used to set up informative crosses which will be analyzed for mineralization phenotypes and scanned for quantitative trait loci by genotyping analysis. The identified candidate genes for modulation of PXE phenotype will be verified by functional assays and by tissue array expression profiles. Our studies will also focus on a specific mutant mouse, Enpp1-/-, which we have recently developed as a model for a severe ectopic mineralization disorder, generalized arterial calcification of infancy, with overlapping clinical features of PXE. The Enpp1-/- mice will be crossed with Abcc6-/- mice, and the developed colonies with double heterozygous as well as homozygous/heterozygous compound mutations will be analyzed for phenotypic modulation. The second area of investigation will focus on identification of consequences of missense mutations in the ABCC6 gene, particularly with respect to loss of functional transporter activity (assayed by an insect cell, Sf9, inside-out vesicle system) and intracellular trafficking of the mutant protein in the liver (as assessed in an in vivo mouse perfusion system). The pathogenic nature of missense mutations will also be verified in a novel zebrafish morpholino knock-down system. Finally, we will attempt the identification of the molecule(s) physiologically transported by ABCC6, by a number of approaches. Collectively, these studies are designed to identify genetic factors that contribute to the pathomechanistic details leading from mutations in the ABCC6 gene to ectopic mineralization of peripheral tissues and to identify specific steps amenable to pharmacologic intervention towards treatment of this, currently intractable disease.
This application proposes to identify genetic factors which contribute to phenotypic variability in pseudoxanthoma elasticum, a heritable ectopic mineralization disorder with considerable morbidity and mortality. The studies are expected to identify specific pathomechanistic pathways that can be interfered by pharmacologic means, thus providing novel treatment modalities for this, currently intractable group of diseases.
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