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

Abstract

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

A Mouse Model of β-Thalassemia Shows a Liver-Specific Down-Regulation of Abcc6 Expression

“…However, there are two major arguments supporting our claims: i) the Abcc6 protein decrease occurred progressively and became only significantly lower than the WT expression in old mice, and ii) the C57BL/6J background in which the Hbb th3/ϩ deletion resides is not as conductive to mineralization as other congenic strains, such as C3H/HeJ, DBA/ 2J, or 129S1/SvJ. 48,49 The latter strains develop severe dystrophic calcification after cardiovascular cryoinjuries, ischemia, or diet stimuli, 50 which is a phenotype referred to as DCC. By examining F2 intercrosses of resistant mice C57BL/6J and susceptible animals C3H/HeJ, Ivandic and colleagues 51 identified a major susceptibility locus (Dyscalc1) on chromosome 7 and additional minor Dyscalc loci (Dyscalc2 to 4) on chromosomes 4, 12, and 14.…”

“…Also, the Hbb th3/ϩ mice could be crossed into the C3H/ HeJ background to verify if combining both the DCC and thalassemia genotypes would lead to a further increase in susceptibility to connective tissue mineralization similar to PXE. From the DCC mice results 48,49 and the present study, it is clear that the level of ABCC6/Abcc6 transport activity in the liver is an influential modulator of calcification that expresses its potential in defined genetic contexts, not only with respect to PXE but also in regard to other pathologies involving ectopic mineralization. It is perhaps for these reasons that PXE manifestations have been reported with high prevalence in ␤-thalassemia patients of Greek and Italian descent.…”

“…Within the Dyscalc1 locus, a single Abcc6 mutation caused a 65% constitutive decrease in Abcc6 protein levels in liver and is directly responsible for the DCC phenotype. 48,52 These three modifier loci that affect the penetrance and expression of the DCC phenotype 50 and the Abcc6 DCC gene variant are all absent in the com-mon C57BL/6J strain, thereby explaining the relative "resistance" of the C57BL/6J to dystrophic mineralization. Interestingly, the DCC mice develop discrete mineralization in several tissues (unpublished data from L.M.…”

Juxta-articular joint-capsule mineralization in CD73 deficient mice: Similarities to patients withNT5Emutations

“…16 This nucleotide variant elicits aberrant splicing of the Abcc6 mRNA, causing deletion of 5 nucleotides at the end of exon 14 and resulting in a frame shift and premature termination codon of translation. 17,18 As a consequence ABCC6 protein levels in the liver are markedly reduced, associated with ectopic mineralization of peripheral connective tissues. Examination of the Abcc6 genomic sequence in the Nt5e ¡/-mice revealed the presence of this SNP (Fig.…”

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].…”