Identification of Abcc6 as the major causal gene for dystrophic cardiac calcification in mice through integrative genomics

Abstract

The genetic factors contributing to the complex disorder of myocardial calcification are largely unknown. Using a mouse model, we fine-mapped the major locus (Dyscalc1) contributing to the dystrophic cardiac calcification (DCC) to an 840-kb interval containing 38 genes. We then identified the causal gene by using an approach integrating genetic segregation and expression array analyses to identify, on a global scale, cis-acting DNA variations that perturb gene expression. By studying two intercrosses, in which the DCC trait segregates, a single candidate gene (encoding the ATPbinding cassette transporter ABCC6) was identified. Transgenic complementation confirmed Abcc6 as the underlying causal gene for Dyscalc1. We demonstrate that in the cross, the expression of Abcc6 is highly correlated with the local mineralization regulatory system and the BMP2-Wnt signaling pathway known to be involved in the systemic regulation of calcification, suggesting potential pathways for the action of Abcc6 in DCC. Our results demonstrate the power of the integrative genomics in discovering causal genes and pathways underlying complex traits.expression quantitative trait locus ͉ transgenic ͉ positional cloning ͉ osteopontin C haracterized by hydroxyapatite deposition in necrotic myocytes, myocardial calcification is common in specific forms of cardiomyopathy and in myocardial infarction. It has been estimated that Ϸ8% of patients with severe myocardial infarction develop myocardial calcification within 6 years, suggesting a genetic predisposition for postinjury healing and remodeling processes (1). Historically, dystrophic cardiac calcification (DCC) has been considered a spontaneous form of cardiomyopathy in mice, associated with a variety of predisposing factors, but with normal blood levels of calcium and phosphate. Experimentally, it can be reproducibly initiated using a transdiaphragmal freeze-thaw injury or a high-phosphorous (HP) diet (2, 3). Several inbred mouse strains, including C3H/HeJ (C3H) and DBA/2J (DBA), are highly susceptible, whereas many other inbred mouse strains, including C57BL/6J (B6), C57BL/10J (B10), A/J, MRL/MpJ, and BALB/cJ are resistant (refs. 4 and 5; X.W., T.A.D., and A.J.L., unpublished data). In DCC susceptible strains, calcification has also been observed in skeletal muscle, including in the tongue and diaphragm, and kidney, suggesting a systemic defect (2, 5).Using quantitative trait locus (QTL) analysis of an F 2 intercross between B6 and C3H mice (BxH), we previously mapped four DCC loci (6, 7). The locus on chromosome 7 (Dyscalc1), which exhibits recessive inheritance, explains 31% of the total genetic variance and is the major contributor. Dyscalc1 was confirmed by separate intercrosses of B6 and DBA mice (BxD) (5, 8). The C3H strain was originally derived from an outbreeding experiment of the DBA strain, leading us to hypothesize that the susceptible strains C3H and DBA share a common diseasecausing allele (8). To fine map the Dyscalc1 locus, we screened a panel of recombinant congenic (RC) s...

An Alternative Splice Variant in Abcc6, the Gene Causing Dystrophic Calcification, Leads to Protein Deficiency in C3H/He Mice

“…We therefore hypothesized that one of these deletions might contribute to the observed deficiency in MRP6 expression. Meng et al (18) previously found no effect of the 10-bp deletion on the mRNA stability, therefore excluding this deletion as causal mutation for DCC.…”

“…MRP6 is mainly expressed in the liver and to a lesser extent in kidney. In contrast, very low levels have been found in tissues affected by dystrophic calcification, such as heart and muscle (18,21,22). Thus, the preventive mechanism of Abcc6 on cardiac calcification seems to be mediated by systemic rather than tissue-specific factors.…”

Latent TGF-β–binding protein 4 modifies muscular dystrophy in mice

“…Dyscalc1 is a major contributor to dystrophic cardiovascular calcification in susceptible strains, affecting both the vessels and myocardium (8). Two genes have been independently implicated in Dyscalc, Abcc6 and Emp3 (9,10). Both genes were excluded as accounting for dMOD1 gene using known disease-associated polymorphisms and finding that both parental strains had the susceptible alleles of each gene (data not shown).…”

Dysregulation of gene expression in ABCC6 knockdown HepG2 cells

“…In addition, deficiency in CD73 (encoded by the NT5E gene) causes a human disease called arterial calcifications due to deficiency in CD73 (ACDC), which is characterized by a spontaneous and premature onset of arterial calcification, closely related but phenotypically different from GACI and PXE [12]. Finally, mutations in the mouse ABCC6 gene have been associated with dystrophic cardiac calcification (DCC), a disease characterized by hydroxyapatite deposition in necrotic myocytes [13,14]. ABCC6 is expressed in the basolateral membrane of hepatocytes and in the proximal kidney tubules [15,16].…”