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C57BL/6J (B6), C57BL/10J (B10), A/J, MRL/MpJ, and BALB/cJ are resistant (refs. 4 and 5; X.W., T.A.D., and A.J.L., unpublished data).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...

Objective ABCC6 genetic deficiency underlies Pseudoxanthoma elasticum (PXE) in humans, characterized by ectopic calcification, and early cardiac disease. The spectrum of PXE has been noted in Abcc6 deficient mice, including dystrophic cardiac calcification. We tested the role of Abcc6 in response to cardiac ischemia-reperfusion (I/R) injury. Methods and results To determine the role of Abcc6 in cardio-protection we induced ischemic injury in mice in vivo by occluding the left anterior descending artery (30min) followed by reperfusion (48hrs). Infarct size was increased in Abcc6 deficient mice compared to wild type controls. Additionally, an Abcc6 transgene significantly reduced infarct size on the background of a naturally occurring Abcc6 deficiency. There were no differences in cardiac calcification following I/R, but increased cardiac apoptosis was noted in Abcc6 deficient mice. Previous studies have implicated the BMP signaling pathway in directing calcification, and here we show the BMP responsive transcription factors, pSmad1/5/8 were increased in hearts of Abcc6 mice. Consistent with this finding, BMP4 and BMP9 were increased, and ALK2 and Endoglin were down-regulated in cardiac extracts from Abcc6 deficient mice versus controls. Conclusions These data identify Abcc6 as a novel modulator of cardiac myocyte survival after I/R. This cardio-protective mechanism may involve inhibition of the BMP signaling pathway, which modulates apoptosis.

Abstract-Previously, a blood pressure (BP) quantitative trait locus (QTL) on rat chromosome 9 (RNO9) was localized to a Ͻ2.4 cM interval using congenic strains generated by introgressing segments of RNO9 from the Dahl salt-resistant (R) rat into the background of the Dahl salt-sensitive (S) rat. Renal gene expression using Affymetrix gene chips was profiled on S and a congenic strain spanning the 2.4-cM BP QTL interval. This analysis identified 20 differentially expressed genes/expressed sequence tags. Of these, the locus with the greatest differential expression (30-to 35-fold) was regulated endocrine-specific protein 18 (Resp18), which also mapped in the 2.4-cM BP QTL interval. Additional substitution mapping located the QTL to Ͻ0.4 cM or Ϸ493 kb. This newly defined QTL region still included Resp18. Nucleotide variants were identified between S and R genomic DNA of Resp18 in the coding, 5Ј regulatory and 3Ј untranslated regions. The coding sequence variation (T/C) occurs in exon 2 and predicts an amino acid change (Ile/Val) in the protein product. Resp18 was considered a differentially expressed positional candidate for the QTL. To fine-map the BP QTL, we constructed a congenic strain with a smaller introgressed region. Compared with the S rat, this strain (1) had significantly lower BP, (2)