Adenovirus-Mediated ABCC6 Gene Therapy for Heritable Ectopic Mineralization Disorders

Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by skin, eye, and cardiovascular lesions due to ectopic mineralization and fragmentation of elastic fibers of connective tissues. We present an atypical case of PXE with diffuse vascular calcification and negligible skin and eye lesions. The patient was a 37-year-old man suffering from severe bilateral arterial calcifications in superficial femoral and posterior tibial arteries. Eye fundoscopy and skin examination were first considered normal. This phenotype suggested first the diagnosis of Arterial Calcification due to Deficiency of CD73 (ACDC) characterized by mutations in NT5E gene. However, we found two variants in ABCC6 gene, and no variant in NT5E. Skin reexamination revealed few lateral skin papules confined to the scalp. Phenotypic overlap was described in vascular calcification disorders, between GACI and PXE phenotypes, and we discuss here expansion of this overlap, including ACDC phenotype. Identification of these expanding and overlapping phenotypes was enabled by genetic screening of the corresponding genes, in a systematic approach. We propose to create a calcification next generation sequencing (NGS) panel with NT5E, GGCX, ENPP1, and ABCC6 genes to improve the molecular diagnosis of vascular calcification.

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Dysregulation of gene expression in ABCC6 knockdown HepG2 cells

Cellular & Molecular Biology Letters ◽

10.2478/s11658-014-0208-2 ◽

2014 ◽

Vol 19 (4) ◽

Cited By ~ 17

Author(s):

Rocchina Miglionico ◽

Maria Armentano ◽

Monica Carmosino ◽

Antonella Salvia ◽

Flavia Cuviello ◽

...

Keyword(s):

Hepg2 Cells ◽

Autosomal Recessive ◽

Pseudoxanthoma Elasticum ◽

Arterial Calcification ◽

Elastic Fibers ◽

Ectopic Calcification ◽

Ectopic Mineralization ◽

Abcc6 Gene ◽

Basolateral Plasma Membrane

AbstractABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.

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Targeted Ablation of the Abcc6 Gene Results in Ectopic Mineralization of Connective Tissues

Molecular and Cellular Biology ◽

10.1128/mcb.25.18.8299-8310.2005 ◽

2005 ◽

Vol 25 (18) ◽

pp. 8299-8310 ◽

Cited By ~ 141

Author(s):

John F. Klement ◽

Yasushi Matsuzaki ◽

Qiu-Jie Jiang ◽

Joseph Terlizzi ◽

Hae Young Choi ◽

...

Keyword(s):

Complex Disease ◽

Soft Tissues ◽

Subcutaneous Tissue ◽

Pseudoxanthoma Elasticum ◽

Connective Tissues ◽

Alizarin Red ◽

Arterial Blood ◽

Ectopic Mineralization ◽

Abcc6 Gene ◽

Total Body

ABSTRACT Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6 −/− mice but was not observed in Abcc6 +/− or Abcc6 +/+ mice up to 2 years of age. A total body computerized tomography scan of Abcc6 −/− mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

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