Analysis of NPPI and ABCC6 function and shared pathogenic pathways in generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE)
Final Report Abstract
Mechanisms of arterial calcification are poorly understood. We identified mutations in ENPP1, encoding for Ectonucleotide pyrophosphatase/ phosphodiesterase family member 1 (NPP1), an enzyme generating extracellular inorganic pyrophosphate (PPi), causing generalized arterial calcification (GACI) in more than 75% of the affected patients. Most recently, we have proven that also mutations in ABCC6, the gene previously associated with pseudoxanthoma elasticum (PXE), account for a subset of cases with GACI. Conversely, mutations in ENPP1 can cause PXE in the absence of ABCC6 mutations. Based on the similarities of GACI and PXE, we hypothesized that the underlying disease genes, ENPP1, ABCC6, drive a cohesive molecular pathophysiology system utilizing common pathways to suppress ectopic tissue mineralization. By studying PPi and ATP concentrations in the media of cultured VSMCs derived from Abcc6 deficient mice, we found that levels of extracellular ATP are regulated by Abcc6 activity and that PPi could be ruled out as a substrate of ABCC6 transport. Interestingly, against our initial hypothesis, downstream regulation of calcification is different in fibroblasts derived from patients carrying ENPP1 and ABCC6 mutations, which might also affect the phenotype of the disease. Furthermore, we found, that fibroblasts derived from GACI patients with ABCC6 mutations have a higher propensity to calcify than fibroblasts derived from PXE patients, which suggests that modifier genes might be involved to cause the severe GACI phenotype instead of the milder PXE phenotype in patients carrying ABCC6 mutations. We analysed the effects of different mutations in ENPP1 patients by transfection of mutant constructs on the functional properties of the enzyme NPP1. All mutations had been found in patients with GACI or had been classified as polymorphisms. We showed that all ENPP1 polymorphisms tested have normal NPP1 function, and we identified an additional functional polymorphism, which had been expected to be pathogenic before. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual NPP1 function. These NPP1 mutants are promising candidates for folding correction and stabilizing of the protein in order to adjust their intracellular trafficking and to normalize their cellular localization. Further in vitro and in vivo studies employing pharmacological chaperones to enhance the enzymatic activity of the mutant proteins are planned.
Publications
- (2012). Generalized arterial calcification of infancy and pseudoxanthoma elasticum: two sides of the same coin. Front Genet. 3:302
Nitschke Y, Rutsch F
(See online at https://doi.org/10.3389/fgene.2012.00302) - (2012).Genetics in arterial calcification: lessons learned from rare diseases.Trends Cardiovasc Med. 22(6):145-9
Nitschke Y, Rutsch F
(See online at https://doi.org/10.1016/j.tcm.2012.07.011) - (2014). Modulators of networks: molecular targets of arterial calcification identified in man and mice. Curr Pharm Des. 20(37):5839-52
Nitschke Y, Rutsch F
(See online at https://doi.org/10.2174/1381612820666140212193330) - (2016). Effects of different variants in the ENPP1 Gene on the functional properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1. Hum Mutat. 2016 Nov;37(11):1190-1201
Stella J, Buers I, van de Wetering K, Höhne W, Rutsch F, Nitschke Y
(See online at https://doi.org/10.1002/humu.23057) - (2016). New perspectives on rare connective tissue calcifying diseases. Curr Opin Pharmacol. 28:14-23
Rashdan NA, Rutsch F, Kempf H, Váradi A, Lefthériotis G, MacRae VE
(See online at https://doi.org/10.1016/j.coph.2016.02.002)
