Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

An Unusual Case of Transient Cardiac Calcification Identified on Antenatal Echocardiography: A Generalized Arterial Calcification of Infancy (GACI) Like Presentation.

Generalised arterial calcification of infancy (GACI) is an ultra-rare life-threatening genetic disorder. Arterial calcification is identified during foetal ultrasound scan (USS) as increased cardiac and/or vascular echogenicity. Inorganic pyrophosphate (PPi) is the main inhibitor of arterial calcification. Pathogenic variants in ENPP1, ABCC6 and NT5E causing low PPi lead to ectopic calcifications. Rheumatoid arthritis (RA) is an acquired condition that can also lead to arterial calcification in adults. We present an extremely rare case of a transient GACI-like condition identified during foetal echocardiogram of an infant born to a mother diagnosed with RA, which spontaneously resolved postnatally. This case highlights that foetal ultrasound scans of pregnant women with RA should be carefully evaluated for cardiovascular calcifications.

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.

A Reference Range for Plasma Levels of Inorganic Pyrophosphate in Children Using the ATP Sulfurylase Method.

PURPOSE: Generalized arterial calcification of infancy, pseudoxanthoma elasticum, autosomal recessive hypophosphatemic rickets type 2, and hypophosphatasia are rare inherited disorders associated with altered plasma levels of inorganic pyrophosphate (PPi). In this study, we aimed to establish a reference range for plasma PPi in the pediatric population, which would be essential to support its use as a biomarker in children with mineralization disorders. METHODS: Plasma samples were collected from 200 children aged 1 day to 18 years who underwent blood testing for medical conditions not affecting plasma PPi levels. PPi was measured in proband plasma utilizing a validated adenosine triphosphate (ATP) sulfurylase method. RESULTS: The analytical sensitivity of the ATP sulfurylase assay consisted of 0.15 to 10 µM PPi. Inter- and intra-assay coefficients of variability on identical samples were below 10%. The standard range of PPi in the blood plasma of children and adolescents aged 0 to 18 years was calculated as 2.36 to 4.44 µM, with a median of 3.17 µM, with no difference between male and female probands. PPi plasma levels did not differ significantly in different pediatric age groups. MAIN CONCLUSIONS: Our results yielded no noteworthy discrepancy to the reported standard range of plasma PPi in adults (2-5 µM). We propose the described ATP sulfurylase method as a diagnostic tool to measure PPi levels in plasma as a biomarker in the pediatric population.

Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies.

Generalized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start-time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Hereditary Disorders of Cardiovascular Calcification.

Arterial calcification is a common phenomenon in the elderly, in patients with atherosclerosis or renal failure and in diabetes. However, when present in very young individuals, it is likely to be associated with an underlying hereditary disorder of arterial calcification. Here, we present an overview of the few monogenic disorders presenting with early-onset cardiovascular calcification. These disorders can be classified according to the function of the respective disease gene into (1) disorders caused by an altered purine and phosphate/pyrophosphate metabolism, (2) interferonopathies, and (3) Gaucher disease. The finding of arterial calcification in early life should alert the clinician and prompt further genetic work-up to define the underlying genetic defect, to establish the correct diagnosis, and to enable appropriate therapy.

Crisponi syndrome/cold-induced sweating syndrome type 2: Reprogramming of CS/CISS2 individual derived fibroblasts into three clones of one iPSC line.

Crisponi syndrome/cold-induced sweating syndrome type 2 (CS/CISS2) is a rare disease with severe dysfunctions of thermoregulatory processes. CS/CISS2 individuals suffer from recurrent episodes of hyperthermia in the neonatal period and paradoxical sweating at cold ambient temperatures in adolescence. Variants in CLCF1 (cardiotrophin-like-cytokine 1) cause CS/CISS2. Here, we summarize the generation of three clones of one stem cell line (iPSC) of a CS/CISS2 individual carrying the CLCF1 variant c.321C>G on both alleles. These patient derived iPSC clones show a normal karyotype, several pluripotency markers, and the ability to differentiate into the three germ layers.

Generation of induced pluripotent stem cell lines from a Crisponi/Cold induced sweating syndrome type 1 individual.

Cytokine receptor like factor 1 (CRLF1) is the gene implicated, when mutated, in Crisponi syndrome/cold-induced sweating syndrome type 1 (CS/CISS1). Here, we report the establishment of induced pluripotent stem cell lines (iPSCs) from fibroblasts of a Turkish CS/CISS1 individual with a homozygous variant in CRLF1 (c.708_709delinsT; p.[Pro238Argfs*6]). This variant is the most frequent variant associated to CS/CISS1 in the Turkish population. These patient derived iPSC lines show all pluripotency markers, a normal karyotype and the ability to differentiate into the three germ layers.

Reversion of arterial calcification by elastin-targeted DTPA-HSA nanoparticles.

Generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE) are characterized by pathologic calcifications in the media of large- and medium sized arteries. GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. Different treatment approaches including bisphosphonates and orally administered pyrophosphate (PPi) were investigated in recent years, but reversion of calcification could not be achieved. With this study, we pursued the idea of a combination of controlled drug delivery through nanoparticles and active targeting via antibody conjugation to develop a treatment for GACI and PXE. To establish a suitable drug delivery system, the chelating drug diethylenetriamine pentaacetic acid (DTPA) was conjugated to nanoparticles composed of human serum albumin (HSA) as biodegradable and non-toxic particle matrix. To accomplish an active targeting of the elastic fibers exposed through calcification of the affected areas, the nanoparticle surface was functionalized with an anti-elastin antibody. Cytotoxicity and cell interaction studies revealed favorable preconditions for the intended i.v. application. The chelating ability was evaluated in vitro and ex vivo on aortic ring culture isolated from two mouse models of GACI and PXE. The positive results led to the conclusion that the produced nanoparticles might be a promising therapy in the treatment of GACI and PXE.

Alkaline Phosphatases Account for Low Plasma Levels of Inorganic Pyrophosphate in Chronic Kidney Disease.

INTRODUCTION: Patients on dialysis and kidney transplant recipients (KTR) present the syndrome of mineral and bone disorders (MBD), which share common traits with monogenic calcifying diseases related to disturbances of the purinergic system. Low plasma levels of inorganic pyrophosphate (PPi) and ectopic vascular calcifications belong to these two conditions. This suggests that the purinergic system may be altered in chronic kidney disease with MBD. Therefore, we perform a transversal pilot study in order to compare the determinants of PPi homeostasis and the plasma levels of PPi in patients on dialysis, in KTR and in healthy people. PATIENTS AND METHODS: We included 10 controls, 10 patients on maintenance dialysis, 10 early KTR 3 ± 1 months after transplantation and nine late KTR 24 ± 3 months after transplantation. We measured aortic calcifications, plasma and urine levels of PPi, the renal fractional excretion of PPi (FePPi), nucleoside triphosphate hydrolase (NPP) and ALP activities in plasma. Correlations and comparisons were assessed with non-parametric tests. RESULTS: Low PPi was found in patients on dialysis [1.11 (0.88-1.35), p = 0.004], in early KTR [0.91 (0.66-0.98), p = 0.0003] and in late KTR [1.16 (1.07-1.45), p = 0.02] compared to controls [1.66 (1.31-1.72) µmol/L]. Arterial calcifications were higher in patients on dialysis than in controls [9 (1-75) vs. 399 (25-526) calcium score/cm2, p < 0.05]. ALP activity was augmented in patients on dialysis [113 (74-160), p = 0.01] and in early KTR [120 (84-142), p = 0.002] compared to controls [64 (56-70) UI/L]. The activity of NPP and FePPi were not different between groups. ALP activity was negatively correlated with PPi (r = -0.49, p = 0.001). DISCUSSION: Patients on dialysis and KTR have low plasma levels of PPi, which are partly related to high ALP activity, but neither to low NPP activity, nor to increased renal excretion of PPi. Further work is necessary to explore comprehensively the purinergic system in chronic kidney disease.

Crisponi/cold-induced sweating syndrome: Differential diagnosis, pathogenesis and treatment concepts.

Crisponi/cold-induced sweating syndrome (CS/CISS) is an autosomal recessive disease characterized by hyperthermia, camptodactyly, feeding and respiratory difficulties often leading to sudden death in the neonatal period. The affected individuals who survived the first critical years of life, develop cold-induced sweating and scoliosis in early childhood. The disease is caused by variants in the CRLF1 or in the CLCF1 gene. Both proteins form a heterodimeric complex that acts on cells expressing the ciliary neurotrophic factor receptor (CNTFR). CS/CISS belongs to the family of "CNTFR-related disorders" showing a similar clinical phenotype. Recently, variants in other genes, including KLHL7, NALCN, MAGEL2 and SCN2A, previously linked to other diseases, have been associated with a CS/CISS-like phenotype. Therefore, retinitis pigmentosa and Bohring-Optiz syndrome-like (KLHL7), Congenital contractures of the limbs and face, hypotonia, and developmental delay syndrome (NALCN), Chitayat-Hall/Schaaf-Yang syndrome (MAGEL2), and early infantile epileptic encephalopathy-11 syndrome (SCN2A) all share an overlapping phenotype with CS/CISS, especially in the neonatal period. This review aims to summarize the existing literature on CS/CISS, focusing on the current state of differential diagnosis, pathogenesis and treatment concepts in order to achieve an accurate and rapid diagnosis. This will improve patient management and enable specific treatments for the affected individuals.

ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP.

Generalized arterial calcification of infancy (GACI) is associated with widespread arterial calcification and stenoses and is caused by mutations in ENPP1. ENPP1 encodes for ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which cleaves ATP to generate inorganic pyrophosphate (PPi) and adenosine monophosphate (AMP) extracellularly. The current study was designed to define the prevalence of arterial stenoses in GACI individuals and to identify the mechanism through which ENPP1 deficiency causes intimal proliferation. Furthermore, we aimed to effectively prevent and treat neointima formation in an animal model of GACI through the systemic administration of recombinant human (rh)ENPP1-Fc protein. Based on a literature review, we report that arterial stenoses are present in at least 72.4% of GACI cases. We evaluated the effect of rhENPP1-Fc on ENPP1-silenced human vascular smooth muscle cells (VSMCs) and on induced intimal proliferation in Enpp1-deficient ttw/ttw mice treated with carotid ligation. We demonstrate that silencing ENPP1 in VSMCs resulted in a tenfold increase in proliferation relative to that of cells transfected with negative control siRNA. The addition of rhENPP1-Fc, AMP or adenosine restored the silenced ENPP1-associated proliferation. In contrast, neither PPi nor etidronate, a current off-label treatment for GACI, had an effect on VSMC proliferation. Furthermore, subcutaneous rhENPP1-Fc protein replacement was effective in preventing and treating intimal hyperplasia induced by carotid ligation in an animal model of GACI. We conclude that ENPP1 inhibits neointima formation by generating  AMP. RhENPP1-Fc may serve as an approach for the effective prevention and treatment of arterial stenoses in GACI.

Endogenous Calcification Inhibitors in the Prevention of Vascular Calcification: A Consensus Statement From the COST Action EuroSoftCalcNet.

The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PPi). The importance of PPi for inhibiting arterial calcification has been reinforced by the protective effects of PPi in various mouse models displaying ectopic calcifications. Besides PPi, Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp-deficient mice develop spontaneous calcification of the arterial media. Whereas PPi and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.

Inherited Arterial Calcification Syndromes: Etiologies and Treatment Concepts.

PURPOSE OF REVIEW: We give an update on the etiology and potential treatment options of rare inherited monogenic disorders associated with arterial calcification and calcific cardiac valve disease. RECENT FINDINGS: Genetic studies of rare inherited syndromes have identified key regulators of ectopic calcification. Based on the pathogenic principles causing the diseases, these can be classified into three groups: (1) disorders of an increased extracellular inorganic phosphate/inorganic pyrophosphate ratio (generalized arterial calcification of infancy, pseudoxanthoma elasticum, arterial calcification and distal joint calcification, progeria, idiopathic basal ganglia calcification, and hyperphosphatemic familial tumoral calcinosis; (2) interferonopathies (Singleton-Merten syndrome); and (3) others, including Keutel syndrome and Gaucher disease type IIIC. Although some of the identified causative mechanisms are not easy to target for treatment, it has become clear that a disturbed serum phosphate/pyrophosphate ratio is a major force triggering arterial and cardiac valve calcification. Further studies will focus on targeting the phosphate/pyrophosphate ratio to effectively prevent and treat these calcific disease phenotypes.

Effects of Different Variants in the ENPP1 Gene on the Functional Properties of Ectonucleotide Pyrophosphatase/Phosphodiesterase Family Member 1.

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E-NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PPi ), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal-recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PPi generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild-type E-NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E-NPP1 enzyme activity were still able to generate PPi and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E-NPP1 function, which would be potential therapeutical targets for conformational-stabilizing agents.

Lmbrd1 expression is essential for the initiation of gastrulation.

The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1). This defect is characterized by massive accumulation of free cobalamin in lysosomes and loss of mitochondrial succinyl-CoA synthesis and cytosolic methionine synthesis. Affected children suffer from heart defects, developmental delay and megaloblastic anemia. LMBRD1 encodes for LMBD1, a predicted lysosomal cobalamin transport protein. In this study, we determine the physiological function of LMBRD1 during embryogenesis by generating Lmbrd1 deficient mice using the Cre/LoxP system. Complete loss of Lmbrd1 function is accompanied by early embryonic death in mice. Whole mount in situ hybridization studies against bone morphogenetic protein 4 and Nodal show that initial formation of the proximal-distal axis is unaffected in early embryonic stages whereas the initiation of gastrulation is disturbed shown by the expression pattern of even skipped homeotic gene 1 and fibroblast growth factor 8 in Lmbrd1 deficient mice. We conclude that intact function of LMBD1 is essential for the initiation of gastrulation.

Novel interferonopathies associated with mutations in RIG-I like receptors.

Type I interferonopathies are a relatively new class of inherited autoimmune disorders associated with an inborn elevated interferon response. Activation of cytosolic receptors which recognize viral double stranded RNA including the RIG-I (retinoic acid-inducible gene I) like receptors RIG-I and MDA5 (melanoma differentiation-associated gene 5) has been shown to induce the transcription of type I interferon genes. Within recent years, with the help of next generation sequencing techniques in syndromic families, mutations in the genes encoding for RIG-I and MDA5 have been identified to cause rare diseases including Aicardi-Goutières syndrome, Systemic Lupus Erythematosus in certain individuals as well as classic and atypical Singleton-Merten syndrome. Patients carrying mono-allelic mutations in MDA5 and RIG-I show constitutive activation of the RIG-I receptors and downstream signalling associated with increased type I interferon production. Although differing in the degree of phenotypic expression and severity, the phenotype of these "novel" diseases shows a considerable overlap reflecting their common pathogenetic pathway.

A specific IFIH1 gain-of-function mutation causes Singleton-Merten syndrome.

Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals' blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption.

Modulators of networks: molecular targets of arterial calcification identified in man and mice.

In recent years, mechanisms of arterial calcifications are beginning to be elucidated. Arterial calcification is now considered as an actively regulated process resembling osteogenesis within the arterial wall orchestrated by a number of systemic or constitutively expressed mediators. Genetic studies of rare monogenic human disorders and studies of naturally occurring or mutant mouse models have identified specific inductors and inhibitors of arterial calcification, which can be classified according to the networks they participate in. These networks include ATP and pyrophosphate metabolism, phosphate homeostasis and vitamin D receptor signaling. Furthermore, intracellular signaling molecules, including SMAD6 and a number of systemic circulatory inhibitors of arterial calcification, including fetuin, tumor necrosis factor receptor superfamily member 11b, matrix GLA protein, adiponectin and family with sequence similarity 20 member A have been identified by human and mouse genetics. Based on the in vivo evidence of their functional relevance, these proteins will serve as excellent targets for the prevention and treatment of arterial calcification. In this review we discuss the functional role of the identified modulators of arterial calcification and describe the networks they belong to.

Generalized arterial calcification of infancy and pseudoxanthoma elasticum: two sides of the same coin.

Generalized arterial calcification of infancy (GACI) is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 (ATP-binding cassette subfamily C number 6) are known to cause pseudoxanthoma elasticum (PXE). However, ABCC6 mutations account for a significant subset of GACI cases, and ENPP1 mutations can also be associated with PXE lesions. Based on the considerable overlap of GACI and PXE, both entities appear to reflect two ends of a clinical spectrum of ectopic calcification rather than two distinct disorders. ABCC6 and ENPP1 mutations might lead to alterations of the same physiological pathways.