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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.

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.

Newborn infants with generalized arterial calcification of infancy are at a substantial risk of sudden cardiac death. Treatment with bisphosphonates while helpful do not guarantee a favorable outcome. Herein we report a case of generalized arterial calcification of infancy from fetal diagnosis through childhood. The patient exhibited previously unreported variants in the ENPP1 gene. Newborn echocardiogram and CT imaging showed extensive calcifications throughout the coronary arteries and aortic arch. Treatment started immediately after birth. Long-term follow-up has demonstrated disease regression. Early recognition and treatment were instrumental in reversing this infant's disease. Successful treatment included the previously reported use of pamidronate but with the novel inclusion of acetazolamide and Similac PM 60/40® (low calcium, low phosphate infant formula).

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.

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.

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.

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.

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.

GACI (generalized arterial calcification of infancy) is a rare autosomal recessive disorder characterized by arterial and periarticular calcifications. Most children die in the first months of life of cardiovascular complications. Hypophosphatemic rickets (HR) resistant to medical treatment may complete the phenotype and is associated with a milder phenotype. This report discusses the case of a girl who presented neonatal ectopic periarticular calcifications with spontaneous regression, and then at the age of 3 years developed HR. There was no clinical improvement after treatment with calcitriol and phosphate, and correction of alkaline phosphatase induced the recurrence of periarticular and tissular calcifications : the treatment was reduced and the bone distortion treated by surgery. GACI diagnosis was confirmed by genetic analysis. At the age of 4.5 years, she developed a retinal abnormality and decreased radial pulse: these clinical signs are usually observed in pseudoxanthoma elasticum (PXE). It is now established that GACI and PXE belong to the same entity characterized by arterial and tissular calcifications of which this original case report is an illustration.

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.

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone.

Pseudoxanthoma elasticum (PXE), CD73 deficiency, generalized arterial calcification of infancy, and progeria involve accelerated medial arterial calcification (MAC) leading to premature cardiovascular morbidity and mortality. MAC is also observed in diabetes mellitus, chronic kidney disease, and the

Inogranic pyrophosphate (PPi) inhibits hydroxyapatite deposition, and mice deficient in the PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) Plasma cell membrane glycoprotein-1 (PC-1) develop peri-articular and arterial calcification in early life. In idiopathic infantile arterial calcification (IIAC), hydroxyapatite deposition and smooth muscle cell (SMC) proliferation occur, sometimes associated with peri-articular calcification. Thus, we assessed PC-1 expression and PPi metabolism in a 25-month-old boy with IIAC and peri-articular calcifications. Plasma PC-1 was