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Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification disorder affecting soft connective tissues that is caused by biallelic ABCC6 mutations. While the underlying pathomechanisms are incompletely understood, reduced circulatory levels of inorganic pyrophosphate (PPi)—a potent mineralization inhibitor—have been reported in PXE patients and were suggested to be useful as a disease biomarker. In this study, we explored the relation between PPi, the ABCC6 genotype and the PXE phenotype. For this, we optimized and validated a PPi measurement protocol with internal calibration that can be used in a clinical setting. An analysis of 78 PXE patients, 69 heterozygous carriers and 14 control samples revealed significant differences in the measured PPi levels between all three cohorts, although there was overlap between all groups. PXE patients had a ±50% reduction in PPi levels compared to controls. Similarly, we found a ±28% reduction in carriers. PPi levels were found to correlate with age in PXE patients and carriers, independent of the ABCC6 genotype. No correlations were found between PPi levels and the Phenodex scores. Our results suggest that other factors besides PPi are at play in ectopic mineralization, which limits the use of PPi as a predictive biomarker for severity and disease progression.

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.

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that cleaves extracellular adenosine triphosphate (ATP) to generate pyrophosphate (PPi), an inorganic metabolite with potent anticalcification activity. Loss-of-function mutations cause hypopyrophosphatemia and lead to a state of ENPP1 deficiency, which has an acute infantile phase known as generalized arterial calcification of infancy (GACI) and a pediatric to adult phase known as autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). ENPP1 deficiency manifests as ectopic calcification of multiple tissues, neointimal proliferation, premature mortality, impaired growth, and bone deformities. INZ-701, a human ENPP1-Fc protein, is in clinical development as an enzyme replacement therapy for the treatment of ENPP1 deficiency. The pharmacokinetic and pharmacodynamic profile and therapeutic effect of INZ-701 were investigated in Enpp1asj/asj mice, a murine model of ENPP1 deficiency. Enpp1asj/asj mice have undetectable plasma PPi, lower plasma phosphate, and higher FGF23 levels compared with wild-type (WT) mice. Enpp1asj/asj mice on the acceleration diet, containing high phosphate and low magnesium, quickly develop clinical signs, including dehydration, rough hair coat, pinned ears, stiffed legs, and hunched back. Enpp1asj/asj mice treated with vehicle had aforementioned clinical signs plus severe ectopic calcification in multiple tissues and bone defects, characteristics of the clinical phenotype observed in GACI and ARHR2 patients. Our results showed a durable PPi response for more than 3 days after a single dose of INZ-701. Treatment of ENPP1-deficient mice every other day with INZ-701 for 8 weeks restored circulating levels of PPi, prevented pathological calcification in all the tested organs, restored growth parameters, corrected bone defects, improved clinical signs, and decreased mortality in Enpp1asj/asj mice, demonstrating the potential of INZ-701 to treat ENPP1 deficiency. © 2021 American Society for Bone and Mineral Research (ASBMR).

Purpose Generalized Arterial Calcification of Infancy (GACI), characterized by vascular calcifications that are often fatal shortly after birth, is usually caused by deficiency of ENPP1. A small fraction of GACI cases result from deficiency of ABCC6, a membrane transporter. The natural history of GACI survivors has not been established in a prospective fashion. Methods We performed deep phenotyping of 20 GACI survivors. Results Sixteen of twenty subjects presented with arterial calcifications, but only 5 had residual involvement at the time of evaluation. Individuals with ENPP1 deficiency either had hypophosphatemic rickets or were predicted to develop it by 14 years of age; 14/16 had elevated intact FGF23 levels (iFGF23). Blood phosphate levels correlated inversely with iFGF23. For ENPP1-deficient individuals, the lifetime risk of cervical spine fusion was 25%, that of hearing loss was 75%, and the main morbidity in adults was related to enthesis calcification. Four ENPP1-deficient individuals manifested classic skin or retinal findings of PXE. We estimated the minimal incidence of ENPP1 deficiency at ~1 in 200,000 pregnancies. Conclusions GACI appears to be more common than previously thought, with an expanding spectrum of overlapping phenotypes. The relationships among decreased ENPP1, increased iFGF23, and rickets could inform future therapies.

PURPOSE OF REVIEW: This review summarizes current understanding of General Arterial Calcification of Infancy (GACI), emphasizing pathophysiology, clinical presentation, and approaches and controversies in management. RECENT FINDINGS: Identification of causative ENPP1 mutations revealed that GACI arises from deficiencies in inorganic pyrophosphate (leading to calcifications) and adenosine monophosphate (leading to intimal proliferation). Identification of genotypic and phenotypic overlap with pseudoxanthoma elasticum and autosomal recessive hypophosphatemic rickets further advanced understanding of GACI as a complex, multisystemic disease. Clinical data is limited to small, retrospective samples; it is therefore unknown whether commonly-used medications, such as bisphosphonates and hypophosphatemia treatment, are therapeutic or potentially harmful. ENPP1-Fc replacement represents a promising approach warranting further study. SUMMARY: Knowledge gaps in natural history place clinicians at high-risk of assigning causality to interventions that are correlated with changes in clinical status. There is thus a critical need for improved natural history studies to develop and test targeted therapies.

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z‐score

Generalized arterial calcification of infancy (GACI) is a rare autosomal-recessive disorder, characterized by calcification of the internal elastic lamina, fibrotic myointimal proliferation of muscular arteries and resultant arterial stenosis. Treatment with bisphosphonates has been proposed as a means of reducing arterial calcifications in GACI patients, although there is no formalized treatment approach. The case reported here was a patient with severe GACI diagnosed at three months of age who had no response to bisphosphonate treatment, but clinically improved after the initiation of magnesium and anti-phosphate (using calcium carbonate) treatments. In patients unresponsive to bisphosphonate, magnesium and anti-phosphate treatment may be attempted.

Loss-of-function mutations in the ABCC6 gene cause pseudoxanthoma elasticum and type 2 generalized arterial calcification of infancy, heritable ectopic mineralization disorders without effective treatment. ABCC6 encodes the putative efflux transporter ABCC6, which is predominantly expressed in the liver. Although the substrate of ABCC6 remains unknown, recent studies showed that pseudoxanthoma elasticum is a metabolic disorder caused by reduced circulating levels of pyrophosphate, a potent mineralization inhibitor. We hypothesized that reconstitution of ABCC6 might counteract ectopic mineralization in an Abcc6–/– mouse model of pseudoxanthoma elasticum. Intravenous administration of a recombinant adenovirus expressing wild-type human ABCC6 in Abcc6–/– mice showed sustained high-level expression of human ABCC6 in the liver for up to 4 weeks, increasing pyrophosphate levels in plasma. In addition, adenovirus injection every 4 weeks restored plasma pyrophosphate levels and, consequently, significantly reduced ectopic mineralization in the skin of young mice. By contrast, the same treatment in old mice with already established mineral deposits failed to reduce mineralization. These results suggest that adenovirus-mediated ABCC6 gene delivery, when initiated early, is a promising prevention therapy for pseudoxanthoma elasticum and generalized arterial calcification of infancy, diseases that currently lack preventive or therapeutic options.

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the “PXE gene” and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue. For this reason, their presence is often considered troublesome in molecular diagnostics. In pseudoxanthoma elasticum (PXE), a disease predominantly caused by mutations in ATP-binding cassette family C member 6 (ABCC6), the presence of two pseudogenes complicates the analysis of sequence data. With whole-exome sequencing (WES) becoming the standard of care in molecular diagnostics, we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6. We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities, contrary to WES. We propose a time- and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE, which is highly automatable.

International audience; Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Overlap has been reported between the inherited PXE phenotype associated with ENPP1, ABCC6 or NT5E mutations and acquired PXE clinical manifestations associated with haemoglobinopathies induced by HBB mutations. No treatment is currently available for PXE. A young boy presented with severe early-onset systemic calcifications occurring in the skin as elastosis perforans serpiginosa (EPS) and in the arteries, causing mesenteric and limb ischaemia. Analyses revealed deleterious ABCC6, ENPP1 and HBB mutations. The diagnosis of severe PXE was retained and we have coined the term 'PXE+ syndrome' to describe the cumulative effects of the various mutations in this uncommon phenotype. Given the severity, rapid progression and a potentially fatal prognosis, intravenous sodium thiosulfate (STS) was initiated at 25 g three times weekly for 6 months. Numerous side-effects prompted dosage adjustment to 10 g intravenously daily. Treatment efficacy was evaluated at 6 months. Asthaenia, anorexia and pre-/postprandial pain had subsided, entailing weight gain. Abdominal EPS had diminished. Calcific stenosis of the coeliac and mesenteric arteries was no longer detectable on arterial ultrasonography. Follow-up revealed only transient efficacy of STS. Discontinuation of treatment to evaluate the persistence of effects resulted in relapse of the initial symptomatology after 4 months. STS efficacy is conceivably due to strong antioxidant properties and chelation of calcium to form soluble calcium thiosulfate complexes. This case is suggestive of PXE+ syndrome for which STS may represent potential treatment in severe cases. What's already known about this topic? Generalized arterial calcification of infancy may occur in association with ABCC6 mutations and pseudoxanthoma elasticum (PXE) can be linked to ENPP1 mutations. A PXE-like phenotype has also been reported in a subset of patients with inherited haemoglobinopathies, namely sickle cell disease or beta-thalassaemia, related to HBB mutations. To date, there is still no cure for PXE. What does this study add? We report a severe case of PXE resulting from the cumulative effects of several deleterious mutations in ENPP1, ABCC6 and HBB. We suggest the term 'PXE+ syndrome' to describe such patients. Sodium thiosulfate therapy could represent a potential option in severe cases of PXE+ syndrome.

we describe the case of an infant hospitalized in our pediatric intensive care unit with suspected miocarditis. The diagnosis of generalized arterial calcification of infancy (GACI) was confirmed after genetic analysis, the patient was 6 days on extracorporeal membrane oxygenation and died after a failed attempt of endovascular stent placement on 19th day of hospitalization. This paper also contains a review of the literature including new insights into genetic and molecular mechanisms concerning the development of GACI, describe the pathophysiology of the disease and discuss the recent possible ways of treatment and management of GACI.

// Qiaoli Li 1 , Joshua Kingman 1 , John P. Sundberg 2 , Michael A. Levine 3 , Jouni Uitto 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA 2 The Jackson Laboratory, Bar Harbor, ME, USA 3 Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: Qiaoli.Li@jefferson.edu Keywords: pseudoxanthoma elasticum, ectopic mineralization, etidronate treatment, bisphosphonates, mouse model Received: March 03, 2016 Accepted: June 09, 2016 Published: July 20, 2016 ABSTRACT Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable disorders manifesting with ectopic tissue mineralization. Most cases of PXE and some cases of GACI are caused by mutations in the ABCC6 gene, resulting in reduced plasma pyrophosphate (PPi) levels. There is no effective treatment for these disorders. It has been suggested that administration of bisphosphonates, stable and non-hydrolyzable PPi analogs, could counteract ectopic mineralization in these disorders. In this study we tested the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6 -/- mice, a model of PXE. The Abcc6 -/- mice received subcutaneous injections of etidronate, 0.283 and 3.40 mg/kg per injection (0.01× and 0.12×), twice a week, in both prevention and reversal studies. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by micro-computed tomography. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as the diagnosis is made, with careful monitoring of potential side effects.

Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare form of hypophosphatemic rickets, which is known to develop in survivors of generalized arterial calcification of infancy (GACI). Both disorders are based on a deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and present with a high clinical variability and a lack of a phenotype-genotype association. ARHR2 is characterized by phosphate wasting due to elevated fibroblast growth factor 23 (FGF23) levels and might represent a response of the organism to minimize ectopic calcification in individuals with ENPP1-deficiency. This report reviews the recent clinical and preclinical data on this ultra-rare disease in childhood.

// Joshua Kingman 1 , Jouni Uitto 1 and Qiaoli Li 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College, and the PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: // Keywords : mouse model, ectopic mineralization, maternal diet, magnesium, generalized arterial calcification of infancy, Pathology Section Received : October 28, 2016 Accepted : March 16, 2017 Published : March 29, 2017 Abstract Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder caused by mutations in the ENPP1 gene. It is characterized by mineralization of the arterial blood vessels, often diagnosed prenatally, and associated with death in early childhood. There is no effective treatment for this devastating disorder. We previously characterized the Enpp1 asj mutant mouse as a model of GACI, and we have now explored the effect of elevated dietary magnesium (five-fold) in pregnant mothers and continuing for the first 14 weeks of postnatal life. The mothers were kept on either control diet or experimental diet supplemented with magnesium. Upon weaning at 4 weeks of age the pups were placed either on control diet or high magnesium diet. The degree of mineralization was assessed at 14 weeks of age by histopathology and a chemical calcium assay in muzzle skin, kidney and aorta. Mice placed on high magnesium diet showed little, if any, evidence of mineralization when their corresponding mothers were also placed on diet enriched with magnesium during pregnancy and nursing. The reduced ectopic mineralization in these mice was accompanied by increased calcium and magnesium content in the urine, suggesting that magnesium competes calcium-phosphate binding thereby preventing the mineral deposition. These results have implications for dietary management of pregnancies in which the fetus is suspected of having GACI. Moreover, augmenting a diet with high magnesium may be beneficial for other ectopic mineralization diseases, including nephrocalcinosis.

Generalized arterial calcification of infancy (GACI) is a rare genetic disease that affects the circulatory system and the large- and medium-sized arteries throughout the body. GACI usually occurs during fetal development. Babies with GACI are diagnosed early, generally soon after birth and in some cases before birth by fetal ultrasound.

// Sarah Y. Siu 1 , Nathaniel A. Dyment 2 , David W. Rowe 2 , John P. Sundberg 3 , Jouni Uitto 1,4 and Qiaoli Li 1,4 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College and The PXE International Center of Excellence in Research and Clinical Care, Thomas Jefferson University, Philadelphia, PA, USA 2 Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CT, USA 3 The Jackson Laboratory, Bar Harbor, ME, USA 4 Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA Correspondence to: Qiaoli Li, email: // Keywords : ectopic mineralization; generalized arterial calcification of infancy; mouse models; cryohistology; Pathology Section Received : October 07, 2016 Accepted : November 02, 2016 Published : November 14, 2016 Abstract Generalized arterial calcification of infancy (GACI) is an autosomal recessive disorder characterized by early onset of extensive mineralization of the cardiovascular system. The classical forms of GACI are caused by mutations in the ENPP1 gene, encoding a membrane-bound pyrophosphatase/phosphodiesterase that hydrolyzes ATP to AMP and inorganic pyrophosphate. The asj-2J mouse harboring a spontaneous mutation in the Enpp1 gene has been characterized as a model for GACI. These mutant mice develop ectopic mineralization in skin and vascular connective tissues as well as in cartilage and collagen-rich tendons and ligaments. This study examined in detail the temporal ectopic mineralization phenotype of connective tissues in this mouse model, utilizing a novel cryo-histological method that does not require decalcification of bones. The wild type, heterozygous, and homozygous mice were administered fluorescent mineralization labels at 4 weeks (calcein), 10 weeks (alizarin complexone), and 11 weeks of age (demeclocycline). Twenty-four hours later, outer ears, muzzle skin, trachea, aorta, shoulders, and vertebrae were collected from these mice and examined for progression of mineralization. The results revealed differential timeline for disease initiation and progression in various tissues of this mouse model. It also highlights the advantages of cryo-histological fluorescent imaging technique to study mineral deposition in mouse models of ectopic mineralization disorders.

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum (PXE), and related conditions with overlapping clinical features include generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate (PPi) and phosphate (Pi), and the findings suggest a unifying pathomechanism relating to reduced PPi/Pi ratio. This hypothesis is based on the notion that PPi serves as a powerful inhibitor of mineralization while Pi is a pro-mineralization factor, and an appropriate PPi/Pi ratio is critical for prevention of ectopic mineralization under homeostatic conditions. PXE International, the premiere patient support organization, advocating on behalf of patients and families with PXE, sponsors regular research meetings evaluating the progress in this and related ectopic mineralization disorders. The latest meetings were held in September 2014 in Bethesda, MD and in September 2015 in Budapest, Hungary. This report summarizes the latest progress in research on PXE and related ectopic mineralization disorders, based on presentations and discussions in these meetings, with pharmacologic implications for currently intractable disorders.

// Jieyu Zhang 1,2 , Nathaniel A. Dyment 3 , David W. Rowe 3 , Sarah Y. Siu 1 , John P. Sundberg 4 , Jouni Uitto 1 and Qiaoli Li 1 1 Department of Dermatology and Cutaneous Biology, The Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA 2 Department of Dermatology, The Fourth Military Medical University, Xijing Hospital, Xi’an, China 3 Center for Regenerative Medicine and Skeletal Development, University of Connecticut Health Center, Farmington, CT, USA 4 The Jackson Laboratory, Bar Harbor, ME, USA Correspondence to: Qiaoli Li, email: // Keywords : ectopic mineralization, tendon and ligament calcification, chondrocalcinosis, generalized arterial calcification of infancy, mouse models, Pathology Section Received : October 22, 2015 Accepted : January 31, 2016 Published : February 17, 2016 Abstract Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. A spontaneous asj-2J mutant mouse has been characterized as a model for GACI. Previous studies focused on phenotypic characterization of skin and vascular tissues. This study further examined the ectopic mineralization phenotype of cartilage, collagen-rich tendons and ligaments in this mouse model. The mice were placed on either control diet or the “acceleration diet” for up to 12 weeks of age. Soft connective tissues, such as ear (elastic cartilage) and trachea (hyaline cartilage), were processed for standard histology. Assessment of ectopic mineralization in articular cartilage and fibrocartilage as well as tendons and ligaments which are attached to long bones were performed using a novel cryo-histological method without decalcification. These analyses demonstrated ectopic mineralization in cartilages as well as tendons and ligaments in the homozygous asj-2J mice at 12 weeks of age, with the presence of immature osteophytes displaying alkaline phosphatase and tartrate-resistant acid phosphatase activities as early as at 6 weeks of age. Alkaline phosphatase activity was significantly increased in asj-2J mouse serum as compared to wild type mice, indicating increased bone formation rate in these mice. Together, these data highlight the key role of ENPP1 in regulating calcification of both soft and skeletal tissues.

Patients with generalized arterial calcification of infancy (GACI) develop vascular calcifications early in life. About half of them die within the first six months despite optimal medical care. A subset of those who survive eventually develop hypophosphatemic rickets. Since hypophosphatemia and hyperphosphaturia have been previously associated with increased survival in GACI patients, physicians often avoid phosphate repletion as treatment for rickets. As a consequence, GACI patients develop severe rachitic complications such as short stature and skeletal deformities. It appears that the recognition of hypophosphatemia later in life in some GACI patients is a consequence of having survived the first few months of life, and not the cause of their survival per se. Here, we report the long-term follow-up of a GACI patient who was phosphate-repleted for his rickets for more than seven years without worsening of vascular calcification.

Pseudoxanthoma elasticum (PXE), the prototype of heritable ectopic mineralization disorders, manifests with deposition of calcium hydroxyapatite crystals in the skin, eyes and arterial blood vessels. This autosomal recessive disorder, due to mutations in ABCC6, is usually diagnosed around the second decade of life. In the spectrum of heritable ectopic mineralization disorders are also generalized arterial calcification of infancy (GACI), with extremely severe arterial calcification diagnosed by prenatal ultrasound or perinatally, and arterial calcification due to CD73 deficiency (ACDC) manifesting with arterial and juxta-articular mineralization in the elderly; the latter disorders are caused by mutations in ENPP1 and NT5E, respectively. The unifying pathomechanistic feature in these three conditions is reduced plasma levels of inorganic pyrophosphate (PPi), a powerful endogenous inhibitor of ectopic mineralization. Several on-going attempts to develop treatments for these conditions, either with the goal to normalize PPi plasma levels or by means of preventing calcium hydroxyapatite deposition independent of PPi, are in advanced preclinical levels or in early clinical trials. This overview summarizes the prospects of treatment development for ectopic mineralization disorders, with PXE, GACI and ACDC as the target diseases, from the 2020 vantage point.

Teeth are comprised of three unique mineralized tissues, enamel, dentin, and cementum, that are susceptible to developmental defects similar to those affecting bone. X-linked hypophosphatemia (XLH), caused by PHEX mutations, leads to increased fibroblast growth factor 23 (FGF23)-driven hypophosphatemia and local extracellular matrix disturbances. Hypophosphatasia (HPP), caused by ALPL mutations, results in increased levels of inorganic pyrophosphate (PP(i)), a mineralization inhibitor. Generalized arterial calcification in infancy (GACI), caused by ENPP1 mutations, results in vascular calcification due to decreased PP(i), later compounded by FGF23-driven hypophosphatemia. In this perspective, we compare and contrast dental defects in primary teeth associated with XLH, HPP, and GACI, briefly reviewing genetic and biochemical features of these disorders and findings of clinical and preclinical studies to date, including some of our own recent observations. The distinct dental defects associated with the three heritable mineralization disorders reflect unique processes of the respective dental hard tissues, revealing insights into their development and clues about pathological mechanisms underlying such disorders.

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., Arterial disease: promising protein replacement therapy in inherited disorder A protein replacement therapy may prove useful in tackling calcification and narrowing of the arteries in babies with a severe genetic disorder. Generalized Arterial Calcification of Infancy (GACI) is a rare condition in which infants’ arteries become calcified and their blood vessels internally scarred. It often leads to congestive heart failure. The ENPP1 gene encodes a protein that is crucial to preventing excess calcium build-up in the body. Mutations in the ENPP1 gene lead to GACI, but no therapies for the condition exist. Now, Frank Rutsch at Muenster University Children’s Hospital in Germany and co-workers have shown that administering a protein replacement can inhibit blood vessel scarring and arterial clogging in GACI mice models and in human stem cell cultures. The protein replacement boosts production of a key metabolic molecule called adenosine monophosphate.

The ATP-binding cassette transporter 6 (ABCC6) gene encodes a cellular transmembrane protein transporter (MRP6) that is involved in the regulation of tissue calcification in mammals. Mutations in ABCC6 are associated with human ectopic calcification disorders. To gain insight into its evolution and involvement in tissue calcification we conducted a comparative analysis of the ABCC6 gene and the related gene ABCC1 from invertebrates to vertebrates where a bony endoskeleton first evolved. Taking into consideration the role of ABCC6 in ectopic calcification of human skin we analysed the involvement of both genes in the regeneration of scales, mineralized structures that develop in fish skin. The ABCC6 gene was only found in bony vertebrate genomes and was absent from Elasmobranchs, Agnatha and from invertebrates. In teleost fish the abcc6 gene duplicated but the two genes persisted only in some teleost genomes. Six disease causing amino acid mutations in human MRP6 are a normal feature of abcc6 in fish, suggesting they do not have a deleterious effect on the protein. After scale removal the abcc6 (5 and 10 days) and abcc1 (10 days) gene expression was up-regulated relative to the intact control skin and this coincided with a time of intense scale mineralization.

BACKGROUND In pseudoxanthoma elasticum (PXE), low pyrophosphate levels may cause ectopic mineralization, leading to skin changes, visual impairment, and peripheral arterial disease.OBJECTIVES The authors hypothesized that etidronate, a pyrophosphate analog, might reduce ectopic mineralization in PXE.METHODS In the Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum trial, adults with PXE and leg arterial calcifications (n = 74) were randomly assigned to etidronate or placebo (cyclical 20 mg/kg for 2 weeks every 12 weeks). The primary outcome was ectopic mineralization, quantified with (18)fluoride positron emission tomography scans as femoral arterial wall target-to-background ratios (TBRfemoral). Secondary outcomes were computed tomography arterial calcification and ophthalmological changes. Safety outcomes were bone density, serum calcium, and phosphate.RESULTS During 12 months of follow-up, the TBRfemoral increased 6% (interquartile range [IQR]: -12% to 25%) in the etidronate group and 7% (IQR: -9% to 32%) in the placebo group (p = 0.465). Arterial calcification decreased 4% (IQR: -11% to 7%) in the etidronate group and increased 8% (IQR: -1% to 20%) in the placebo group (p = 0.001). Etidronate treatment was associated with significantly fewer subretinal neovascularization events (1 vs. 9, p = 0.007). Bone density decreased 4% -12% in the etidronate group and 6% -9% in the placebo group (p = 0.374). Hypocalcemia (1.5 mmol/l) and recovered spontaneously.CONCLUSIONS In patients with PXE, etidronate reduced arterial calcification and subretinal neovascularization events but did not lower femoral 18fluoride sodium positron emission tomography activity compared with placebo, without important safety issues. (Treatment of Ectopic Mineralization in Pseudoxanthoma elasticum; NTR5180) (c) 2018 by the American College of Cardiology Foundation.

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), which normally hydrolyzes extracellular ATP into AMP and pyrophosphate (PPi). The disease is characterized by extensive arterial calcification and stenosis of large- and medium-sized vessels, leading to vascular-related complications of hypertension and heart failure. There is currently no effective treatment available, but bisphosphonates – nonhydrolyzable PPi analogs – are being used off-label to reduce arterial calcification, although this has no reported impact on the hypertension and cardiac dysfunction features of GACI. In this study, the efficacy of a recombinant human ENPP1 protein therapeutic (rhENPP1) was tested in Enpp1asj-2J homozygous mice (Asj-2J or Asj-2J hom), a model previously described to show extensive mineralization in the arterial vasculature, similar to GACI patients. In a disease prevention study, Asj-2J mice treated with rhENPP1 for 3 weeks showed >95% reduction in aorta calcification. Terminal hemodynamics and echocardiography imaging of Asj-2J mice also revealed that a 6-week rhENPP1 treatment normalized elevated arterial and left ventricular pressure, which translated into significant improvements in myocardial compliance, contractility, heart workload and global cardiovascular efficiency. This study suggests that ENPP1 enzyme replacement therapy could be a more effective GACI therapeutic than bisphosphonates, treating not just the vascular calcification, but also the hypertension that eventually leads to cardiac failure in GACI patients., Summary: ENPP1 enzyme replacement therapy can have important implications for generalized arterial calcification of infancy by treating both vascular calcification and hypertension, which are the leading causes of cardiac failure and mortality in patients.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) was originally reported as a responsible gene for generalized arterial calcification in infancy (GACI). Though the prognosis of GACI patients is poor because of myocardial infarction and heart failure in relation to medial calcification of the coronary arteries, some patients rescued by bisphosphonate treatment have been reported. Recently, ENPP1 is also reported as responsible for autosomal recessive hypophosphatemic rickets type 2. We show here a boy with homozygous ENPP1 mutations diagnosed as having GACI in early infancy. After the diagnosis, he was treated with etidronate disodium (EHDP) in combination with antihypertensive drugs. The calcification of major arteries was diminished and disappeared by the age of eight months. He also showed mild hypophosphatemia (2.6–3.7 mg/dl) from the age of one year. After the treatment with EHDP for five years, he showed genu valgum with hypophosphatemia (2.6 mg/dl). He was diagnosed as having hypophosphatemic rickets at the age of seven years. The findings that hyper-mineralization of the arteries and hypo-mineralization of the bone observed in the same patient are noteworthy. ENPP1 could be regarded as a controller of the calcification of the whole body at least in part., Highlights • A boy with homozygous ENPP1 mutation suffered GACI and subsequent hypophosphatemic rickets. • ENPP1 mutation caused both hyper-mineralization in the arteries and hypo-mineralization in the bone in the same patient. • ENPP1 could be regarded as a mineralization controller of the body.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6 −/− mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6 −/− mice over several months prevented the development of pseudoxanthoma elasticum–like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6 −/− mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

The presence of calcification in atherosclerotic arteries has long been recognized, but the mechanisms of calcification remain poorly understood. Risk factors of atherosclerosis, such as age, sex, hyperlipidemia, diabetes mellitus, hypertension, left ventricular hypertrophy, and smoking, have been shown to predict future coronary events. Traditionally, parameters known to be of predictive value were included in conventional risk scores such as the Framingham risk score 1 and helped to divide patients into high risk, intermediate risk, and low risk of future cardiovascular events. 2 Also, many studies have shown that calcification is accelerated in the presence of diabetes mellitus and chronic renal failure. 3–5 Since the introduction of computed tomography (CT), a noninvasive technology with the ability to not only determine the extent of coronary narrowing but also determine the extent of vessel wall thickening, remodeling, and, above all, the presence or absence of calcification was lacking. Agatston et al 6 devised a coronary artery calcification (CAC) scoring scheme, and others have shown calcium score to be a better predictor of future events than the Framingham risk index alone. 7 There is also a close relationship between the presence of CAC and atherosclerotic plaque burden, with angiographic studies showing high sensitivity but poor specificity of CAC score for predicting obstructive disease. 8 Physical activity is known to decrease cardiovascular mortality; however, there is no relationship between the extent of coronary calcification as assessed by CT in individuals engaged in moderate exercise. 9 Allison et al 10 showed that calcification in different vascular bed was predictive of different outcomes. Coronary calcification was predictive of cardiovascular disease mortality, whereas calcification in thoracic aorta, carotid arteries, and iliac arteries were predictive of total mortality when followed for 7.8 years. 10 At the same time, histopathologic studies indicate that heavily calcified plaques are stable plaques and unlikely to lead to coronary events, whereas the vulnerable plaque tends to be either noncalcified or with only mild to moderate calcification, suggesting that calcification may exert a protective effect. 11 These studies indicate that calcification is a complex, organized, and highly regulated process. Demer and Tintut 12 have suggested that we divide calcification into 3 categories, that is, inflammatory (atherosclerotic, mostly intimal), metabolic (chronic kidney disease [CKD] and diabetes mellitus, mostly medial), and genetic background (pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcifications attributable to deficiency in CD73, and Marfan syndrome, mostly medial), although admitting that this may be an oversimplification. We will discuss the mechanisms of intimal atherosclerotic and valvular calcification, as well as calcification secondary to metabolic disorders because of some similarity in risk factors and clinical relevance.

SituationA 30 week gestation male weighing 1.66kg presented with metabolic acidosis and high lactate and subsequently developed heart failure and hypertension. He initially started enteral feeds but these were later not tolerated and TPN commenced. On day 8 calcification of the aorta was identified on echocardiogram. CT scans showed extensive arterial calcification including the thoracic and abdominal aorta, subclavian and common carotid arteries, coeliac axis, SMA, renal arteries and iliac vessels. Generalised arterial calcification of infancy (GACI) due to ENPP1 mutation was suspected.BackgroundGACI, a rare autosomal recessive condition can be caused by ENPP1 mutation leading to low levels of inorganic pyrophosphate (PPi), a negative regulator of calcification. GACI has a high mortality rate, up to 55% at 6 months. Mortality has been shown to improve in those who survive the first few months of life.1TreatmentIntravenous sodium thiosulfate, licensed for cyanide poisoning and used off-label for calciphylaxis in adults,2 was commenced to try and reduce existing calcification. Dosing that has been known to be used in three other babies from two different centres,3 was used - 12.5g/m2 over 30minutes on alternate days for 2 weeks followed by 12.5g/m2 five days a week. This is in the same scale as adult calciphylaxis dosing and up to 400mg/kg can be used in paediatric cyanide poisoning. Bisphosphonates were commenced to prevent further calcification. Etidronate, a non-nitrogen containing bisphosphonate, was preferred due to its closer structural similarity to PPi than second generation bisphosphonates. Etidronate has been discontinued in the UK so was not initially available and a dose of pamidronate was given. A Canadian import of etidronate was sourced and commenced a week later. Due to SMA and coeliac axis calcification there were concerns regarding bowel perfusion and he was TPN fed except for 20ml/kg/day EBM. Etidronate 20mg/kg/day was commenced in three divided doses to improve gastrointestinal tolerance.OutcomeInitially his heart failure stabilised and hypertension managed with carvedilol. By day 35 full enteral feeds were reached and he was breathing unassisted in air. CT after one month’s treatment showed no worsening of vascular calcification, though unfortunately calcification did not appear to have improved. At 7 weeks he became tachypnoeic due to worsening heart failure and required respiratory support. Despite ongoing medical therapies he passed away at 8 weeks of age.Challenges and lessons learntDue to the rarity of the condition information on treatment options, dosing and monitoring are limited and the need to use an imported product lead to a short delay in treatment. Etidronate is only available in tablet form but Didronel brand can be crushed and suspended in water,4 Information about the suspension’s uniformity is unavailable but due to a lack of alternatives this was the option taken. A two hour break either side of etidronate while recommended, was compromised to ninety minutes as he required three hourly feeds. Combination treatment was used to try to reduce the calcification; however the extent of calcification had already caused significant cardiac compromise which ultimately led to his demise.ReferencesFerreira C, Ziegler S, Gahl WA. Generalized Arterial Calcification of Infancy. 2014 In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews [Online]. Seattle (WA): University of Washington, Seattle; 1993–2018. Available from: https://www.ncbi.nlm.nih.gov/books/NBK253403Nigwekar SU, Brunelli SM, Meade D, et al. Sodium thiosulfate therapy for calcific uremic arteriolopathy. Clin J Am Soc Nephrol, 2013;8:1162–70.Personal communication: Medicines Information, Alder Hey Children’s NHS Foundation Trust. Email sent to: Leeds Medicines Advisory Service. 28th June 2018.White R, Bradnam V. Handbook of Drug Administration via Enteral Feeding Tubes, Third ed. Cornwall: Pharmaceutical Press; 2015. www.medicinescomplete.com (accessed August 2018).

Pseudoxanthoma elasticum (PXE) is characterized by aberrant mineralization of connective tissues, causing considerable morbidity and mortality. The disease is typically of late onset, the skin manifestations first being noted in the teens or later. Another aberrant mineralization disorder, generalized arterial calcification of infancy (GACI), is present at birth and can demonstrate a phenotypic overlap with PXE.A patient with PXE was noted to have skin findings as early as at 6 years of age, with cardiovascular involvement. The purpose of this study was to examine the genetic basis of this phenotypic presentation in the spectrum of PXE/GACI.The patient's genotype was studied by sequencing ABCC6 and ENPP1, genes known to be associated with PXE and/or GACI.Screening of the ABCC6 gene revealed two pathogenetic mutations, p.R1141X and g.del23-29. Analysis of the ENPP1 gene failed to demonstrate the presence of mutations.This study demonstrates the presence of cutaneous findings of PXE in an 8-year-old paediatric patient, with cardiovascular involvement, illustrating the phenotypic spectrum of PXE.

Ectopic mineralization has been linked to several common clinical conditions with considerable morbidity and mortality. The mineralization processes, both metastatic and dystrophic, affect the skin and vascular connective tissues. There are several contributing metabolic and environmental factors that make uncovering of the precise pathomechanisms of these acquired disorders exceedingly difficult. Several relatively rare heritable disorders share phenotypic manifestations similar to those in common conditions, and, consequently, they serve as genetically controlled model systems to study the details of the mineralization process in peripheral tissues. This overview will highlight diseases with mineral deposition in the skin and vascular connective tissues, as exemplified by familial tumoral calcinosis, pseudoxanthoma elasticum, generalized arterial calcification of infancy, and arterial calcification due to CD73 deficiency. These diseases, and their corresponding mouse models, provide insight into the pathomechanisms of soft tissue mineralization and point to the existence of intricate mineralization/anti-mineralization networks in these tissues. This information is critical for understanding the pathomechanistic details of different mineralization disorders, and it has provided the perspective to develop pharmacological approaches to counteract the consequences of ectopic mineralization.

Summary Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder, is characterized by early mineralization of blood vessels, often diagnosed by prenatal ultrasound and usually resulting in demise during the first year of life. It is caused in most cases by mutations in the ENPP1 gene, encoding an enzyme that hydrolyzes ATP to AMP and inorganic pyrophosphate, the latter being a powerful anti-mineralization factor. Recently, a novel mouse phenotype was recognized as a result of ENU mutagenesis - those mice developed stiffening of the joints, hence the mutant mouse was named 'ages with stiffened joints' (asj). These mice harbor a missense mutation, p.V246D, in the Enpp1 gene. Here we demonstrate that the mutant ENPP1 protein is largely absent in the liver of asj mice, and the lack of enzymatic activity results in reduced inorganic pyrophosphate (PPi) levels in the plasma, accompanied by extensive mineralization of a number of tissues, including arterial blood vessels. The progress of mineralization is highly dependent on the mineral composition of the diet, with significant shortening of the lifespan on a diet enriched in phosphorus and low in magnesium. These results suggest that the asj mouse can serve as an animal model for GACI.

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 preserve transport 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.

Pseudoxanthoma elasticum is a prototype of heritable ectopic mineralization disorders, with phenotypic overlap with generalized arterial calcification of infancy and arterial calcification due to CD73 deficiency. Recent observations have suggested that the reduced inorganic pyrophosphate/phosphate ratio is the cause of soft connective tissue mineralization in these disorders. PXE International, a patient advocacy organization, supports research in part by sponsoring biennial research symposia on these disorders; the latest meeting was held in September 2016 at Thomas Jefferson University, Philadelphia. This report summarizes the progress in pseudoxanthoma elasticum and other ectopic mineralization disorders, as presented in the symposium, with focus on translational aspects of precision medicine toward improved diagnostics and treatment development for these currently intractable disorders.

Background: Arterial stenosis leading to hypertension or heart failure is common in patients with Generalized Arterial Calcification of Infancy (GACI), an ultra-rare disease associated with loss of function mutations in ENPP1, an ectonucleotide pyrophosphatase that hydrolyzes extracellular ATP. GACI is characterized by accelerated calcification and severe myointimal proliferation. The role of ENPP1 in myoinitimal proliferation is unknown. Here, we examined the effect of ENPP1 on proliferation of human vascular smooth muscle cells (hVSMCs). Methods: ENPP1 expression was assessed in primary hVSMCs using qRT-PCR. ENPP1 expression was silenced using siRNA and its activity was verified by a cell based assay or inorganic pyrophosphate (PPi) assay and its effect on VSMC proliferation was determined by 3H-thymidine incorporation. Results: Treatment of primary hVSMCs with siRNA specific to the hENPP1 resulted in ~90% decrease in ENPP1 levels. Cellular enzyme activity correlated with ENPP1 expression in VSMCs. Silencing ENPP1 in hVSMCs led to 1-3 fold increase in proliferation relative to that of cells transfected with control siRNA in 2 out of 2 donors. Peak cell proliferation was observed at 5 days post-transfection. Human iPSC derived VSMCs (iVSMCs) expressed higher levels of ENPP1 than primary hVSMCs. Silencing ENPP1 in iVSMCs resulted in 3-5 fold increase in proliferation relative to that of cells transfected with negative control siRNA in 2 out of 2 donors. Addition of recombinant ENPP1-Fc protein restored ENPP1- associated proliferation in all donors (8099.75 ±134.32 (untreated) vs 1478 ± 55.34 (5ug/ml ENPP1-Fc treated, P Conclusion: For the first time, we have demonstrated that ENPP1 knockdown promotes proliferation of human VSMCs, and treatment with a functionally active ENPP1-Fc protein significantly inhibits ENPP1-associated proliferation. These results suggest that ENPP1 enzyme replacement may be a potential strategy to treat myointimal proliferation in patients with GACI disease.

Generalized arterial calcification of infancy (GACI) is a life-threatening disorder in young infants. Cardiovascular symptoms are usually apparent within the first month of life. The symptoms are caused by calcification of large and medium-sized arteries, including the aorta, coronary arteries, and renal arteries. Most of the patients die by 6 months of age because of heart failure. Recently, homozygous or compound heterozygous mutations for the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene were reported as causative for the disorder. ENPP1 regulates extracellular inorganic pyrophosphate (PP(i)), a major inhibitor of extracellular matrix calcification. A newborn was diagnosed with GACI. The infant died at the age of 7 weeks of cardiac failure and the parents were referred to Molecular Biology and Cytogenetic lab for further workup. Cytogenetics analysis was performed on the parents, which showed normal karyotypes and mutational analysis for the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene was also performed. The mutational analysis showed that both father and mother of the deceased infant were heterozygous carriers of the mutation c.749C>T (p.P250L) in exon 7 of ENPP1 and it was likely, that the deceased child carried the same mutation homozygous on both alleles and died of GACI resulting from this ENPP1 mutation. The couple was counseled and monitored for the second pregnancy. Amniocentesis was performed at 15 weeks of gestation for mutational analysis of the same gene in the second pregnancy. The analysis was negative for the parental mutations. One month after the birth of a healthy infant, peripheral blood was collected from the baby and sent for reconfirmation. The results again were negative for the mutation and the baby was on 6 months follow up and no major symptoms were seen. The parents of the child benefited enormously by learning about the disease much in advance and also its risk of recurrence. The main aim of this study is to emphasize on two aspects: (i) the importance of modern molecular techniques in diagnosis such a syndrome and (2) the difficulties faced by the physician to provide appropriate diagnosis and the adequate genetic counseling to the family without molecular facilities.