Olivier Le Saux

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

A Spectrum of ABCC6 Mutations Is Responsible for Pseudoxanthoma Elasticum

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

The Distribution of Abcc6 in Normal Mouse Tissues Suggests Multiple Functions for this ABC Transporter

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.

Acquired Pseudoxanthoma elasticum-like syndrome in β-thalassaemia patients

Summary. A significant number of patients diagnosed with β‐thalassaemia develop clinical and histopathological manifestations similar to those of an inherited disorder called Pseudoxanthoma elasticum (PXE). The inherited PXE is caused by mutations in the ATP‐binding cassette, subfamily C (CFTR/MRP), member 6 (ABCC6) gene and is characterized by mineralized elastic fibres in dermal, vascular and ocular tissues. As no disease‐causing variant was found in the ABCC6 gene of 10 β‐thalassaemia patients with a PXE‐like phenotype, the present study suggests that the PXE‐like symptoms in these β‐thalassaemic patients are not related to ABCC6 mutations.

Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.

Heterozygosity for a single mutation in the ABCC6 gene may closely mimic PXE: consequences of this phenotype overlap for the definition of PXE.

OBJECTIVES To illustrate a phenotypic overlap consisting of usual, but limited, or atypical manifestations of pseudoxanthoma elasticum (PXE) between heterozygous carriers of a single ABCC6 mutation and patients diagnosed with PXE, carriers of homozygous or compound heterozygous mutations. DESIGN Evaluation for full and typical, incomplete, mild, or overlooked PXE during a 5-year period (2001-2005) based on the following 1992 expert consensus conference items: (1) yellowish papular skin eruption, (2) dermal elastorrhexis and mineralization of elastic fibers in lesional skin, and (3) angioid streaks. Testing for ABCC6 mutations was performed in all cases after informed consent. SETTING French multidisciplinary outpatient clinic for patients with PXE. PARTICIPANTS Patients prospectively referred for PXE and first-degree relatives. Main Outcome Measure Prevalence of PXE with a limited or atypical phenotype and manifesting heterozygosity. RESULTS Ninety-four patients were diagnosed as having PXE. Fifty-eight relatives were also examined, and none displayed the characteristic signs of the disease. Despite the histoclinical items and ABCC6 genotyping, we were unable to establish a definite diagnosis in 5 additional referred cases, ie, to distinguish between PXE with a limited or atypical phenotype and heterozygosity with skin and/or ophthalmologic and/or cardiovascular manifestations suggestive of PXE. CONCLUSIONS We assume that all categories established at the 1992 consensus conference correspond to PXE, but that the 5 patients reported herein also have PXE. Homozygous, compound heterozygous, or heterozygous individuals may fulfill only some of the clinical and/or histopathologic consensus criteria of PXE. They cannot be placed into any category. Expressivity is highly variable in carriers of 1 or 2 ABCC6 mutations, and the disease manifestations overlap between both genotypes. Physicians should thus be more cautious with respect to the prognosis when faced with heterozygous relatives of a patient diagnosed with undisputable PXE. Indeed, heterozygotes may uncommonly experience severe ophthalmologic complications. Whether they may also have cardiovascular complications related to or worsened by PXE remains to be determined.

Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6-/- mice. Abcc6-/- mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.

The vascular phenotype in Pseudoxanthoma elasticum and related disorders: contribution of a genetic disease to the understanding of vascular calcification.

Vascular calcification is a complex and dynamic process occurring in various physiological conditions such as aging and exercise or in acquired metabolic disorders like diabetes or chronic renal insufficiency. Arterial calcifications are also observed in several genetic diseases revealing the important role of unbalanced or defective anti- or pro-calcifying factors. Pseudoxanthoma elasticum (PXE) is an inherited disease (OMIM 264800) characterized by elastic fiber fragmentation and calcification in various soft conjunctive tissues including the skin, eyes, and arterial media. The PXE disease results from mutations in the ABCC6 gene, encoding an ATP-binding cassette transporter primarily expressed in the liver, kidneys suggesting that it is a prototypic metabolic soft-tissue calcifying disease of genetic origin. The clinical expression of the PXE arterial disease is characterized by an increased risk for coronary (myocardial infarction), cerebral (aneurysm and stroke), and lower limb peripheral artery disease. However, the structural and functional changes in the arterial wall induced by PXE are still unexplained. The use of a recombinant mouse model inactivated for the Abcc6 gene is an important tool for the understanding of the PXE pathophysiology although the vascular impact in this model remains limited to date. Overlapping of the PXE phenotype with other inherited calcifying diseases could bring important informations to our comprehension of the PXE disease.

ABCC6 is a basolateral plasma membrane protein

Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. Conclusions: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.

Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa.

Abstract. Pseudoxanthoma elasticum (PXE) is a heritable elastic tissue disorder recently shown to be attributable to mutations in the ABCC6 (MRP6) gene. Whereas PXE has been identified in all ethnic groups studied to date, the prevalence of this disease in various populations is uncertain, although often assumed to be similar. A notable exception however is the prevalence of PXE among South African Afrikaners. A previous report has suggested that a founder effect may explain the higher prevalence of PXE in Afrikaners, a European-derived population that first settled in South Africa in the 17th century. To investigate this hypothesis, we performed haplotype and mutational analysis of DNA from 24 South African families of Afrikaner, British and Indian descent. Among the 17 Afrikaner families studied, three common haplotypes and six different disease-causing variants were identified. Three of these mutant alleles were missense variants, two were nonsense mutations and one was a single base-pair insertion. The most common variant accounted for 53% of the PXE alleles, whereas other mutant alleles appeared at lower frequencies ranging from 3% to 12%. Haplotype analysis of the Afrikaner families showed that the three most frequent mutations were identical-by-descent, indicating a founder origin of PXE in this population.