Mutations In ABCC6 Research Articles

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6–/– Mice

Soft-tissue calcification is associated with aging, common conditions such as diabetes or hypercholesterolemia, and with certain genetic disorders. ABCC6 is an efflux transporter primarily expressed in liver facilitating the release of adenosine triphosphate from hepatocytes. Within the liver vasculature, adenosine triphosphate is converted into pyrophosphate, a major inhibitor of ectopic calcification. ABCC6 mutations thus lead to reduced plasma pyrophosphate levels, resulting in the calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Most mutations in ABCC6 are missense, and many preservetransport activity but are retained intracellularly. We have previously shown that the chemical chaperone 4-phenylbutyrate (4-PBA) promotes the maturation of ABCC6 mutants to the plasma membrane. In a humanized mouse model of pseudoxanthoma elasticum, we investigated whether 4-PBA treatments could rescue the calcification inhibition potential of selected ABCC6 mutants. We used the dystrophic cardiac calcification phenotype of Abcc6-/- mice as an indicator of ABCC6 function to quantify the effect of 4-PBA on human ABCC6 mutants transiently expressed in the liver. We showed that 4-PBA administrations restored the physiological function of ABCC6 mutants, resulting in enhanced calcification inhibition. This study identifies 4-PBA treatment as a promising strategy for allele-specific therapy of ABCC6-associated calcification disorders.

Pseudo-xanthome élastique : une cause rare d’hémorragies digestives chez l’enfant

Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive connective tissue disorder, characterized by calcification and progressive fragmentation of elastic fibers. Gastrointestinal lesions are rare in children and may be responsible for serious complications. This article reports two pediatric cases of PXE revealed by gastrointestinal bleeding. An 11-year-old boy and a 12-year-old girl were hospitalized for gastrointestinal bleeding. Digestive endoscopy showed hemorrhagic gastroenteritis bulbitis in the first case and it was normal in the second. Abdominal ultrasound showed diffuse linear calcifications in both cases. The diagnosis of PXE was retained based on the presence of vascular disease in both patients, a skin lesion in the girl, and an ocular lesion in the boy. The genetic study confirmed the diagnosis of PXE identifying two ABCC6 mutations in the composite state in the boy: the c.2263G> A (p.G755R) mutation on exon 18 and the c.3421C> T (pR1141X) mutation on exon 24 and the 4021G> A (R1164Q) mutation in the homozygous state of ABCC6 exon 24 in the girl. Digestive manifestations are unusual; however, pseudoxanthoma elasticum should be considered in all cases of gastrointestinal bleeding for no apparent reason. Early diagnosis allows prevention and measures to control the risk factors and limit the progression of complications.

Abstract 17417: ABCC6 Deficiency Promotes Atherosclerosis

Introduction: Vascular calcification is associated with aging and common conditions such as hypercholesterolemia and certain genetic disorders. A molecular pathway inhibiting ectopic calcification is initiated by ABCC6, a membrane transporter primarily expressed in liver. ABCC6 facilitates the cellular efflux of ATP from hepatocytes, which is rapidly converted into pyrophosphate (PPi) in the liver vasculature. ABCC6 is responsible for the majority of the PPi release from the liver, and ~60% of plasma PPi in both in humans and mice. As PPi is a major inhibitor of calcification, mutations in ABCC6 underlie the currently incurable calcification disorder pseudoxanthoma elasticum (PXE), some cases of generalized arterial calcification of infancy (GACI). Hypothesis: Multiple lines of evidence have suggested a pathological synergy between ABCC6 and hyperlipidemia. Methods: We examined crosses between Abcc6-/- and Ldlr-/- mice and serum samples from human PXE patients. Three mouse genotypes were generated Ldlr-/-, Ldlr-/-xAbcc6-/- and Ldlr-/- xAbcc6+/-. Results: When subjected to a high fat diet, mice with reduced or no Abcc6 expression displayed a 2-fold increase of atherosclerotic plaque area (p=0.0001). Interestingly, atherosclerotic plaque was also found in Abcc6-deficient mice fed a normal diet but not in control Ldlr-/- animals. Aortic calcification was not significantly affected by Abcc6 expression except in Ldlr-/-xAbcc6-/- mice fed regular chow (p=0.0001). We observed an increased proliferation of smooth muscle cells of but no increase in monocyte-macrophage infiltration in the plaque of Ldlr-/-xAbcc6-/- mice. Importantly, we determined that the cholesterol efflux from isolated macrophages towards HDL (mediated by ABCG1) was significantly decreased (-28%, p=0.006) in Ldlr-/-xAbcc6-/- mice while the efflux towards ApoA1 (mediated by ABCA1) was unchanged. The analysis of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions in these mice showed altered profiles similar to those found with human PXE plasma samples. Conclusions: Our results indicated that, in addition to being an important inhibitor of vascular calcification, ABCC6 inhibits atherogenesis by modulating reverse cholesterol transport.

Cyclosporine A Treatment Inhibits Abcc6-Dependent Cardiac Necrosis and Calcification following Coxsackievirus B3 Infection in Mice.

Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

Genetic Heterogeneity of Pseudoxanthoma Elasticum: The Chinese Signature Profile of ABCC6 and ENPP1 Mutations

Pseudoxanthoma elasticum (PXE), an autosomal recessive disorder characterized by ectopic mineralization, is caused by mutations in the ABCC6 gene. We examined clinically 29 Chinese PXE patients from unrelated families, so far the largest cohort of Asian PXE patients. In a subset of 22 patients, we sequenced ABCC6 and another candidate gene, ENPP1, followed by pathogenicity analyses for each variant. We identified a total of 17 distinct mutations in ABCC6, 15 of them being previously unreported, including 5 frame-shift and 10 missense variants. In addition, a missense mutation in combination with a recurrent nonsense mutation in ENPP1 was discovered in a pediatric PXE case. No cases with p.R1141X or del23-29 mutations, common in Caucasian patient populations, were identified. The 10 missense mutations in ABCC6 were expressed in mouse liver via hydrodynamic tail-vein injections. One mutant protein showed cytoplasmic accumulation indicating abnormal subcellular trafficking, while the other nine mutants showed correct plasma membrane location. These nine mutations were further investigated for their pathogenicity using a recently developed zebrafish mRNA rescue assay. Minimal rescue of the morpholino-induced phenotype was achieved with 8 of the 9 mutant human ABCC6 mRNAs tested, implying pathogenicity. This study demonstrates that the Chinese PXE population harbors unique ABCC6 mutations. These genetic data have implications for allele-specific therapy currently being developed for PXE.

Vitamin K reduces hypermineralisation in zebrafish models of PXE and GACI.

The mineralisation disorder pseudoxanthoma elasticum (PXE) is associated with mutations in the transporter protein ABCC6. Patients with PXE suffer from calcified lesions in the skin, eyes and vasculature, and PXE is related to a more severe vascular calcification syndrome called generalised arterial calcification of infancy (GACI). Mutations in ABCC6 are linked to reduced levels of circulating vitamin K. Here, we describe a mutation in the zebrafish (Danio rerio) orthologue abcc6a, which results in extensive hypermineralisation of the axial skeleton. Administration of vitamin K to embryos was sufficient to restore normal levels of mineralisation. Vitamin K also reduced ectopic mineralisation in a zebrafish model of GACI, and warfarin exacerbated the mineralisation phenotype in both mutant lines. These data suggest that vitamin K could be a beneficial treatment for human patients with PXE or GACI. Additionally, we found that abcc6a is strongly expressed at the site of mineralisation rather than the liver, as it is in mammals, which has significant implications for our understanding of the function of ABCC6.

Large-scaled metabolic profiling of human dermal fibroblasts derived from pseudoxanthoma elasticum patients and healthy controls.

Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.

ABCC6- a new player in cellular cholesterol and lipoprotein metabolism?

BackgroundDysregulations in cholesterol and lipid metabolism have been linked to human diseases like hypercholesterolemia, atherosclerosis or the metabolic syndrome. Many ABC transporters are involved in trafficking of metabolites derived from these pathways. Pseudoxanthoma elasticum (PXE), an autosomal-recessive disease caused by ABCC6 mutations, is characterized by atherogenesis and soft tissue calcification.MethodsIn this study we investigated the regulation of cholesterol biosynthesis in human dermal fibroblasts from PXE patients and healthy controls.ResultsGene expression analysis of 84 targets indicated dysregulations in cholesterol metabolism in PXE fibroblasts. Transcript levels of ABCC6 were strongly increased in lipoprotein-deficient serum (LPDS) and under serum starvation in healthy controls. For the first time, increased HMG CoA reductase activities were found in PXE fibroblasts. We further observed strongly elevated transcript and protein levels for the proprotein convertase subtilisin/kexin type 9 (PCSK9), as well as a significant reduction in APOE mRNA expression in PXE.ConclusionIncreased cholesterol biosynthesis, elevated PCSK9 levels and reduced APOE mRNA expression newly found in PXE fibroblasts could enforce atherogenesis and cardiovascular risk in PXE patients. Moreover, the increase in ABCC6 expression accompanied by the induction of cholesterol biosynthesis supposes a functional role for ABCC6 in human lipoprotein and cholesterol homeostasis.

Mutations in the ABCC6 Gene as a Cause of Generalized Arterial Calcification of Infancy: Genotypic Overlap with Pseudoxanthoma Elasticum

Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by congenital calcification of large and medium sized arteries, associated with early myocardial infarction, heart failure, and stroke, and premature death. Most cases of GACI are caused by mutations in the ENPP1 gene. We first studied two siblings with GACI from a non-consanguineous family without mutations in the ENPP1 gene. To search for disease-causing mutations, we identified genomic regions shared between the two affected siblings but not their unaffected parents or brother. The ABCC6 gene, which is mutated in pseudoxanthoma elasticum (PXE), resided within a small region of homozygosity shared by the affected siblings. Sequence analysis of ABCC6 revealed that the two affected siblings were homozygous for the missense mutation p.R1314W. Subsequently, ABCC6 mutations were identified in five additional GACI families with normal ENPP1 sequences. Genetic mutations in ABCC6 in patients with PXE are associated with ectopic tissue mineralization in the skin and arterial blood vessels. Thus, our findings provide additional evidence that the ABCC6 gene product inhibits calcification under physiologic conditions and confirm a second locus for GACI. In addition, our study emphasizes the potential utility of shared homozygosity mapping to identify genetic causes of inherited disorders.

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Can APOE and MTHFR polymorphisms have an influence on the severity of cardiovascular manifestations in Italian Pseudoxanthoma elasticum affected patients?

BackgroundThe clinical phenotype of Pseudoxanthoma elasticum (PXE) affected patients, although progressive with age, is very heterogeneous, even in the presence of identical ABCC6 mutations, thus suggesting the occurrence of modifier genes. Beside typical skin manifestations, the cardiovascular (CV) system, and especially the peripheral vasculature, is frequently and prematurely compromised. Methods and resultsA cohort of 119 Italian PXE patients has been characterized for apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR. The severity of the clinical phenotype has been quantified according to the Phenodex PXE International score system. Statistical analysis (chi2 test, odd ratio, regression analysis, analysis of variance) were done by GraphPad. Data demonstrate that the frequency of APOE alleles is similar in PXE patients and in healthy subjects and that the allelic variant E2 confers a protection against the age-related increase of CV manifestations. By contrast, PXE patients are characterized by high frequency of the MTHFR-T677T polymorphism. With age, CV manifestations in T677T, but also in C677T, patients are more severe than those associated with the C677C genotype. Interestingly, compound heterozygosity for C677T and A1298C polymorphisms is present in 70% of PXE patients. ConclusionsPXE patients may be screened for these polymorphisms in order to support clinicians for a better management of disease-associated CV complications.

Dysregulation of gene expression in ABCC6 knockdown HepG2 cells.

ABCC6 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.

Premature Termination Codon Read-Through in the ABCC6 Gene: Potential Treatment for Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder manifesting with ectopic connective tissue mineralization, caused by mutations in the ABCC6 gene, ~35% of all mutations being premature termination mutations. In this study, we investigated the therapeutic potential of the nonsense codon read-through-inducing drug, PTC124, in treating PXE. The ability of this drug to facilitate read-through of nonsense mutations was examined in HEK293 cells transfected with human ABCC6 expression constructs harboring seven different PXE associated nonsense mutations, and evaluated by immunofluorescence and In-Cell ELISA. Our data demonstrated that PTC124 did not exhibit cell toxicity in concentrations up to 40 µg/ml, and the facilitated read-through was not only dose dependent but also sequence context dependent. Considering the redundancy of the genetic code, it was postulated that in case of the most common recurrent nonsense mutation, p.R1141X, the read-through may result in substitution of the arginine 1141 by either glycine, tryptophan or cysteine. Their potential pathogenicity was tested in a recently developed zebrafish mRNA rescue assay, and demonstrated that all three mRNA transcripts were able to rescue abcc6a morpholino-induced phenotype of zebrafish. Thus, our results suggest that read-through of nonsense mutations in ABCC6 by PTC124 may have potential for pharmacologic treatment of PXE.

Atorvastatin counteracts aberrant soft tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6 −/− )

Pseudoxanthoma elasticum (PXE), a multisystem heritable disorder with aberrant mineralization of arterial blood vessels, is caused by mutations in the ABCC6 gene. Previous studies have suggested that carriers of the ABCC6 mutations, particularly of p.R1141X, are at increased risk for coronary artery disease. In this study, we used Abcc6 (tm1Jfk) knock-out mice to determine the serum lipid profiles and examine the effects of atorvastatin on the aberrant mineralization in this model of PXE. First, serum lipid profiles at 12 weeks of age, after overnight fasting, revealed a statistically significant increase in total cholesterol and triglyceride levels in Abcc6 (tm1Jfk) mice compared to their wild-type littermates. Placing these mice at 4 weeks of age for 20 weeks on atorvastatin, either 0.01 % or 0.04 % of the diet (low statin and high statin groups, respectively), reduced the total triglyceride and cholesterol levels, which was accompanied with significantly reduced mineralization of the dermal sheath of vibrissae, a biomarker of the aberrant mineralization process in these mice. However, if the mice were placed on atorvastatin for 12 weeks at 12 weeks of age, at which time point significant mineralization had already taken place, no difference in the amount of mineralization was noted. These observations suggest that statins, particularly atorvastatin, can prevent, but not reverse, aberrant mineralization in this mouse model of PXE. For a clinical perspective, a survey of 1,747 patients with PXE was conducted regarding their present or past use of statins. The results indicated that about one third of all PXE patients are currently or have previously been on cholesterol-lowering drugs. Thus, a sizable number of patients with PXE could be subject to modulation of their mineralization processes by concomitant statin treatment. The Abcc6 (-/-) mice serve as a model system for PXE, an ectopic mineralization disorder Abcc6 (-/-) mice were shown to have elevated serum cholesterol and triglyceride levels Feeding of the Abcc6 (-/-) mice with atorvastatin prevented connective tissue mineralization A third of patients with PXE was found to be on cholesterol-lowering therapy Atorvastatin may potentially be beneficial for patients with PXE.

Is classical pseudoxanthoma elasticum a consequence of hepatic ‘intoxication’ due to ABCC6 substrate accumulation in the liver?

Pseudoxanthoma elasticum (PXE) is a serious genetic disorder with ectopic mineralization affecting the skin, the eye and the cardiovascular system. The disease is predominantly caused by mutations in the transmembrane ABC protein ABCC6, a putative small substrate transporter. Interestingly, ABCC6 seems virtually absent in the affected organs, whereas a high expression is seen in hepatocytes. This and further published experimental evidence indicate that PXE is a systemic, metabolic liver disease where circulatory changes affect the peripheral mineralization process. Owing to the well-characterized transport of organic substrates by related ABC proteins, it has been proposed that PXE is caused by impaired export of an antimineralization compound to the blood. The authors here present an alternative hypothesis that explains ectopic mineralization in PXE as a consequence of hepatic accumulation of ABCC6 substrate(s) that via gene-regulating effects leads to altered hepatic secretion and activation of antimineralization/anticalcification proteins such as fetuin-A and Gla proteins.

Clinical Phenotypes and ABCC6 Gene Mutations in Brazilian Families with Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE; OMIM 264800) manifests with characteristic skin lesions of yellowish papules which coalesce into plaques of inelastic and leathery skin on the predilection sites (1). The ocular findings consist of angioid streaks, choroidal neovascularization and subretinal hemorrhages resulting in loss of visual acuity and occasional blindness. Cardiovascular problems include hypertension, intermittent claudication, and occasional myocardial infarcts and stroke. The prevalence of PXE is estimated to be in the range of 1:50,000–70,000 and to be more frequent in females than in males. The diagnosis can be challenging to clinicians due to late-onset of clinical manifestations and considerable heterogeneity. PXE is associated with mutations in the ABCC6 gene which encodes a transmembrane efflux transporter expressed primarily in the liver and the kidneys (2). Consequently, PXE has been suggested to be a metabolic disorder with ectopic mineralization of the peripheral connective tissues. Recent studies have also suggested that cutaneous features of PXE can be found in patients with generalized arterial calcification of infancy due to mutations in the ENPP1 gene (3, 4). Previous studies, which have documented close to 600 distinct mutations in the ABCC6 gene, have suggested the presence of unique mutations affecting certain ethnic groups with different ancestral backgrounds (5, 6). In this study, we asked the specific question whether Brazilian patients of mixed European, Native Indian and African ancestry with PXE harbor unique ABCC6 mutations, and whether such mutations might be correlated with the clinical phenotypes in this population with particular reference to heterozygous carriers.

Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruch's membrane

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of connective tissue, which leads to pathology in eye, skin and blood vessels. The disease is caused by mutations in ABCC6. To learn more about PXE eye pathology, we analyzed Bruch's membrane (BM) of the eye of an Abcc6 knockout mouse. With age, BM differences between Abcc6−/− and wild type mice became apparent. At two years of age, von Kossa staining indicated clear calcification of BM in Abcc6−/− mice, and not in healthy controls. Electron microscopy revealed BM changes as early as at 10 months of age: Fibrous structures with abnormal high electron-density were present in the central layers of BM of Abcc6−/− mice. EDX (Energy Dispersive X-ray) analysis demonstrated that these structures contained elevated levels of Ca, P and O. Since some of these electron-dense structures showed a banding pattern with periodicity of about 50 nm, they most likely represent calcified collagen fibers. Immunoelectron microscopy showed that the calcified structures were positive for collagen III. Remarkably, the elastic layer of BM appeared to have a normal ultrastructure, even in 2.5 year old Abcc6−/− mice. Our results suggest that Abcc6 deficiency in the mouse causes calcification of BM. While PXE is considered to affect primarily the elastic fibers, we found predominantly mineralization of collagen fibers.

The molecular and physiological roles of ABCC6: more than meets the eye.

Abnormal mineralization occurs in the context of several common conditions, including advanced age, diabetes, hypercholesterolemia, chronic renal failure, and certain genetic conditions. Metabolic, mechanical, infectious, and inflammatory injuries promote ectopic mineralization through overlapping yet distinct molecular mechanisms of initiation and progression. The ABCC6 protein is an ATP-dependent transporter primarily found in the plasma membrane of hepatocytes. ABCC6 exports unknown substrates from the liver presumably for systemic circulation. ABCC6 deficiency is the primary cause for chronic and acute forms of ectopic mineralization described in diseases such as pseudoxanthoma elasticum (PXE), β-thalassemia, and generalized arterial calcification of infancy (GACI) in humans and dystrophic cardiac calcification (DCC) in mice. These pathologies are characterized by mineralization of cardiovascular, ocular, and dermal tissues. PXE and to an extent GACI are caused by inactivating ABCC6 mutations, whereas the mineralization associated with β-thalassemia patients derives from a liver-specific change in ABCC6 expression. DCC is an acquired phenotype resulting from cardiovascular insults (ischemic injury or hyperlipidemia) and secondary to ABCC6 insufficiency. Abcc6-deficient mice develop ectopic calcifications similar to both the human PXE and mouse DCC phenotypes. The precise molecular and cellular mechanism linking deficient hepatic ABCC6 function to distal ectopic mineral deposition is not understood and has captured the attention of many research groups. Our previously published work along with that of others show that ABCC6 influences other modulators of calcification and that it plays a much greater physiological role than originally thought.

Generalized Arterial Calcification of Infancy and Pseudoxanthoma Elasticum Can Be Caused by Mutations in Either ENPP1 or ABCC6

Spontaneous pathologic arterial calcifications in childhood can occur in generalized arterial calcification of infancy (GACI) or in pseudoxanthoma elasticum (PXE). GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. However, the genetic basis in subsets of both disease phenotypes remains elusive. We hypothesized that GACI and PXE are in a closely related spectrum of disease. We used a standardized questionnaire to retrospectively evaluate the phenotype of 92 probands with a clinical history of GACI. We obtained the ENPP1 genotype by conventional sequencing. In those patients with less than two disease-causing ENPP1 mutations, we sequenced ABCC6. We observed that three GACI patients who carried biallelic ENPP1 mutations developed typical signs of PXE between 5 and 8 years of age; these signs included angioid streaks and pseudoxanthomatous skin lesions. In 28 patients, no disease-causing ENPP1 mutation was found. In 14 of these patients, we detected pathogenic ABCC6 mutations (biallelic mutations in eight patients, monoallelic mutations in six patients). Thus, ABCC6 mutations account for a significant subset of GACI patients, and ENPP1 mutations can also be associated with PXE lesions in school-aged children. Based on the considerable overlap of genotype and phenotype of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification and other organ pathologies, rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways in tissues beyond the artery.

ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: Relevance to pathomechanisms of pseudoxanthoma elasticum

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6 −/− mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6 −/− animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum.