291 Functional characterization of ABCC6 missense variants implicated in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene. The majority of mutations in ABCC6 are missense variants causing a single amino acid substitution which can result in loss-of-function of ABCC6 protein through changes in its transport activity, cellular trafficking, or conformational stability. As ABCC6 has a specialized efflux function in the liver by contributing to plasma PPi levels, we characterized the potential pathogenicity of six missense variants via intravenous administration of recombinant adenovirus expressing the human ABCC6 cDNA carrying each variant to the liver of Abcc6-/- mouse model of PXE. The consequences of the variants were compared to the wild type protein expressed by an adenovirus which was previously shown to reconstitute ABCC6 in the basolateral plasma membrane of hepatocytes, the physiologic location for ABCC6. Expression of the wild type protein raised plasma levels of PPi, and consequently counteracted the ectopic calcification phenotype in Abcc6-/- mice. In contrast, variant p.L420V showed mixed plasma membrane and cytoplasmic expression, failed to normalize plasma PPi levels and had no effects on ectopic calcification. Variant p.R1138W showed very little expression while p.T364R was expressed mostly intracellularly in the liver, suggesting their pathogenicity. In contrast, p.R391G, p.S400F, and p.R760W variants are less likely to be pathogenic mutations since they resulted in expression similar to the wild type protein, restored plasma PPi levels, and prevented ectopic calcification in the Abcc6-/- mice. These results suggest that adenovirus-mediated liver-specific ABCC6 expression in Abcc6-/- mice can be used as an experimental system to elucidate the functional consequences of human ABCC6 missense variants identified in PXE patients.