This is a revised renewal application for the grant, Biochemistry and Morphology of Connective Tissue (R01 AR28450), which has been active since April, 1981. This project focuses on pseudoxanthoma elasticum (PXE), the prototype of Mendelian autosomal recessive disorder characterized by ectopic mineralization of connective tissues in a variety of organs, with considerable morbidity and mortality. Based on the extensive progress made over the past 10 years of support, we now plan to extend our investigation to define the molecular events that lead to phenotypic expression of PXE, in the spectrum of other heritable disorders with ectopic mineralization, particularly generalized arterial calcification of infancy (GACI) and arterial calcification with CD73 deficiency (ACDC) caused by mutations in the ABCC6, ENPP1 and NT5E genes, respectively. These studies will primarily focus on Abcc6-/- Enpp1asj-2J and Nt5e-/- mice which recapitulate the clinical, genetic and histopathologic features of PXE, GACI and ACDC, respectively. It is now clear that there is a complex pro- mineralization/anti-mineralization network, and mutations in specific genes can result in hydroxyapatite crystal deposition in these disorders, with phenotypic and genotypic overlap. Recent data by us and others have documented alterations in this network, with particular focus on the role of inorganic phosphate (Pi)/inorganic pyrophosphate (PPi) ratio which is critical to prevent ectopic mineralization under physiologic homeostasis.
The Specific Aims propose interconnected investigations on PXE, GACI and ACDC.
Specific Aim 1 proposes expansion of the PXE mutation database towards finding novel genes and new mutations both by candidate gene and next generation sequencing approaches. The novel aspect of this study is to focus on potential mutations in the genes encoding enzymes and transporter systems regulating Pi/PPi metabolism in tissues. This study will also extend the mutation analysis to previously unexplored ethnic populations.
Specific Aim 2 delineates the pro-mineralization/anti-mineralization pathways by assay of enzymes and transporter proteins participating in regulation of PPi/Pi ratio in the liver and skin of Abcc6-/-, Enpp1asj-2J and Nt5e-/- mice by quantitative PCR, Western analysis, and by enzymatic assays. The consequences of the absence of ABCC6 activity, beyond the pathway involving Pi/PPi, will be examined by global microarray and proteomics analyses.
Specific Aim 3 proposes a new area of development of therapies for PXE, GACI and ACDC using Abcc6-/-, Enpp1asj-2J and Nt5e-/- mice as preclinical platforms by testing the effects of pyrophosphate analogues, bisphosphonates, to counteract ectopic mineralization. Collectively, we anticipate that these approaches will disclose critical pathomechanistic details explaining the consequences of ABCC6, ENPP1 and NT5E gene mutations at the phenotypic level, and identification of specific pathways leading to ectopic mineralization will provide information serving as a preclinical platform for development of translational strategies to counteract these devastating, currently intractable, multi-system disorders.
Pseudoxanthoma elasticum (PXE), generalized arterial calcification of infancy (GACI), and arterial calcification with CD73 deficiency (ACDC) are heritable ectopic mineralization disorders with considerable phenotypic and genotypic overlap. This project is continuation of our studies to identify the pathomechanistic pathways leading from genetic mutations into the clinical presentations. The experiments are designed to provide preclinical information in mouse models of these diseases, with implications for development of treatment modalities for these, currently intractable, disorders.
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