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 ATP-binding 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.

Fig. 1.

Identification of Abcc6 as a candidate gene for the Dyscalc1 locus. (A) QPCR analysis of Abcc6 expression in liver, heart, and kidney (n = four mice each strain fed with chow diet; ∗, P < 0.05; †, P < 0.0001). Expression values for B6 homozygotes were set as 100. (B) Western blot analysis showing greatly reduced ABCC6 protein levels in liver in the DBA and C3H strains as compared with HcB24 and B6 strains.

Fig. 2.

Confirmation of Abcc6 as the causal gene for Dyscalc1 using transgenic complementation strategy. (A) One B6-originated BAC clone was selected based on its coverage on chromosome 7 (closed boxes). The whole BAC, containing Abcc6 and two additional genes (BC013491 and Nomo1), was used to construct transgenic mice TgB. After restriction enzyme (Mlu I) digestion, the main fragment of the BAC containing only the Abcc6 gene was used to build transgenic mice TgA. (B) Confirmation of Abcc6 expression in transgenic mice. Hepatic tissue QPCR analysis of five strains are shown (n = four mice, each strain fed with HP diet for 4 weeks). Expression levels are normalized to the mean expression of Abcc6 observed in the F₁ (BxH) mice. (C) DCC phenotypic characterization of all strains. Average values are indicated. F, female; M, male; Tg, transgenic; TgCtrl, nontransgenic littermate. The numbers of mice used for each group are in parentheses. ∗, P < 0.05.

Fig. 3.

Abcc6 is coregulated with local calcification regulators and genes from the BMP2-Wnt pathway. (A) Abcc6 transcript levels in liver and muscle microarray data sets are significantly associated with Abcc6 allelic genotype, but in opposite directions. The BxH F₂ mice were subdivided into three genotypic groups at the Abcc6 locus. Relative expression values were calculated from mean log ratio (“mlratio”; see SI Methods), and all values were adjusted to positive values. Expression of the homozygotes with the highest mlratio value was set as 100. (B) QPCR results for genes from skeletal muscle between the Abcc6 transgenic and littermate control mice fed with HP diet for 4 weeks. Relative expression values are compared with transcript levels in Abcc6 transgenic (100). ∗, P < 0.05; †, P < 0.0001.

Fig. 4.

Expression of Abcc6 is associated with decreased systemic OPN response. (A) QPCR results showed after HP diet treatment that there is an up-regulation of Spp1 gene in liver, heart, aorta, and muscle tissues in the C3H mice but not in Abcc6 transgenic mice. Relative expression values are compared with transcript levels in Abcc6 transgenics (100). P values are indicated. (B) A plasma OPN surge was observed for C3H mice after the mice were fed an HP diet for 7 days. The surge was completely abolished in Abcc6 transgenic mice. ∗, P < 0.05.