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ENPP1/CD203a is a membrane-bound ectonucleotidase capable of hydrolyzing ATP, cGAMP and other substrates. Its enzymatic activity plays an important role in the balance of extracellular adenine nucleotides and the modulation of purinergic signaling, in soft tissue calcification, and in the regulation of the cGAS/STING pathway. However, a detailed analysis of ENPP1 surface expression on human immune cells has not been performed. Here, we selected VHH domains from human ENPP1-immunized alpacas to generate heavy-chain antibodies targeting ENPP1, and analyzed cell surface expression on all circulating immune cell subsets using flow cytometry. We find high expression of ENPP1 in CD141 high conventional dendritic cells (cDC1), while ENPP1 was not detectable on other dendritic cells and monocytes. In the lymphocytic compartment, only CD56 bright natural killer cells and mucosal-associated invariant T cells (MAIT) express ENPP1. In contrast, all other T cell subpopulations, CD56 dim natural killer cells and B lymphocytes do not or only minimally express ENPP1. In summary, we describe highly cell type-specific expression of ENPP1 in the immune system using a newly generated heavy-chain antibody. This reagent will help to decipher the function of ENPP1 in the regulation of the immune response, allow a quick identification of ENPP1-deficiency and of ENPP1-positive tumors, and constitutes the basis for targeted anti-tumor intervention.

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.

Background ENPP1 Deficiency—caused by biallelic variants in ENPP1— leads to widespread arterial calcification in early life (Generalized Arterial Calcification of Infancy, GACI) or hypophosphatemic rickets in later life (Autosomal Recessive Hypophosphatemic Rickets type 2, ARHR2). A prior study using the Exome Aggregation Consortium (ExAC)—a database of exomes obtained from approximately 60,000 individuals—estimated the genetic prevalence at approximately 1 in 200,000 pregnancies. Methods We estimated the genetic prevalence of ENPP1 Deficiency by evaluating allele frequencies from a population database, assuming Hardy–Weinberg equilibrium. This estimate benefitted from a comprehensive literature review using Mastermind ( https://mastermind.genomenon.com/ ), which uncovered additional variants and supporting evidence, a larger population database with approximately 140,000 individuals, and improved interpretation of variants as per current clinical guidelines. Results We estimate a genetic prevalence of approximately 1 in 64,000 pregnancies, thus more than tripling the prior estimate. In addition, the carrier frequency of ENPP1 variants was found to be highest in East Asian populations, albeit based on a small sample. Conclusion These results indicate that a significant number of patients with ENPP1 Deficiency remain undiagnosed. Efforts to increase disease awareness as well as expand genetic testing, particularly in non-European populations are warranted, especially now that clinical trials for enzyme replacement therapy, which proved successful in animal models, are underway.

Generalized arterial calcification of infancy (GACI) is an extremely rare, often fatal condition characterized by widespread calcification and stenosis of both large and medium-sized arteries. The majority of cases are caused by biallelic variants in the ENPP1 gene, leading to deficiency of an enzyme critical for preventing pathological calcium deposition in arterial walls. The prognosis of GACI is severe, with fetal and infant mortality rates reaching up to 55%. This study reports a GACI case diagnosed at 24 weeks in a 32-year-old woman with a history of miscarriages, whose fetal ultrasound at 24 weeks revealed striking echogenic foci and thickening in the heart and major blood vessels. This prompted a multidisciplinary team to suspect GACI. Following confirmation through amniocentesis and given the poor prognosis, the parents opted to terminate the pregnancy at 26 weeks. This case underscores the critical role of advanced prenatal imaging and genetic analysis in diagnosing GACI. Ultimately, management decisions extend beyond medical science, involving profound ethical and personal considerations for families facing this devastating diagnosis.

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) plays a critical role by converting extracellular ATP to AMP, generating extracellular PPi, a potential inhibitor of calcification. Pathogenic variants in the ENPP1 cause generalized arterial calcification of infancy (GACI [OMIM 208000]). GACI, is an ultra-rare disease characterized by early-onset calcification of large and medium-sized arteries, leading to severe cardiovascular complications such as heart failure, pulmonary stenosis (PS), hypertension, and more. In this study, we report a novel homozygous splice-site pathogenic variant in ENPP1 (NM_006208, c.2230 + 5G > A; p.Asp701Asnfs*2) residing in C-terminal nuclease-like domain (NLD) of ENPP1 protein in a Pakistani family diagnosed with severe valvular PS and mild right ventricular hypertrophy (RVH). cDNA assays confirmed the skipping of exon 21, and the splice product underwent nonsense-mediated decay. Functional studies on fibroblasts from the patient demonstrated increased calcification and decreased enzymatic activity of ENPP1, recapitulating the hallmarks of GACI. By combining genetic analysis with the in vitro study, we substantiate that ENPP1 :c.2230 + 5G > A variant is pathogenic, underscoring its role in the development of GACI.

Generalized arterial calcification of infancy (GACI) is a rare, autosomal recessive disorder caused by pathogenic variants in ENPP1 or ABCC6. While typically fatal in infancy, survival into childhood is increasingly recognized. We report a family with 3 affected siblings homozygous for an ENPP1 variant (c.1367G  >  A, p.Arg456Gln). The oldest died in infancy, the surviving 2 received early bisphosphonate therapy. Both survivors demonstrate persistent vascular calcifications, early-onset pseudoxanthoma elasticum (PXE)-like skin lesions, and chronic hypophosphatemia without radiographic rickets. Uniquely, this report contrasts the clinical course of a late preterm sibling against a sibling born extremely premature. Additional findings include auricular cartilage, renal and retinal calcifications, highlighting the systemic nature of ENPP1 deficiency. Our report expands the phenotypic spectrum of ENPP1-related GACI.

Generalized Arterial Calcification of Infancy (GACI) is a rare autosomal recessive disorder characterized by pathological calcium deposition in large and medium-sized arteries, leading to severe cardiovascular complications such as hypertension, heart failure, and stroke. The mortality rate is approximately 50% within the first six months of life if untreated. The disease is primarily caused by mutations in the ENPP1 or ABCC6 genes, resulting in a deficiency of inorganic pyrophosphate (PPi), a key inhibitor of arterial calcification. This review provides a comprehensive overview of the pathophysiology, genetic basis, and clinical features of GACI. In addition, we summarize current and emerging therapeutic strategies, including enzyme replacement therapy with recombinant ENPP1 (INZ-701), critically discussing available preclinical and early clinical evidence, as well as current limitations.

Pseudoxanthoma Elasticum (PXE) is an inherited disease characterized by elastic fiber calcification in the eyes, the skin and the cardiovascular system. PXE results from mutations in ABCC6 that encodes an ABC transporter primarily expressed in the liver and kidneys. It took nearly 15 years after identifying the gene to better understand the etiology of PXE. ABCC6 function facilitates the efflux of ATP, which is sequentially hydrolyzed by the ectonucleotidases ENPP1 and CD73 into pyrophosphate (PPi) and adenosine, both inhibitors of calcification. PXE, together with General Arterial Calcification of Infancy (GACI caused by ENPP1 mutations) as well as Calcification of Joints and Arteries (CALJA caused by NT5E/CD73 mutations), forms a disease continuum with overlapping phenotypes and shares steps of the same molecular pathway. The explanation of these phenotypes place ABCC6 as an upstream regulator of a purinergic pathway (ABCC6 → ENPP1 → CD73 → TNAP) that notably inhibits mineralization by maintaining a physiological Pi/PPi ratio in connective tissues. Based on a review of the literature and our recent experimental data, we suggest that PXE (and GACI/CALJA) be considered as an authentic “purinergic disease”. In this article, we recapitulate the pathobiology of PXE and review molecular and physiological data showing that, beyond PPi deficiency and ectopic calcification, PXE is associated with wide and complex alterations of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease.

Generalized arterial calcification of infancy (GACI) is a rare genetic disorder with high infantile mortality, described to be due to ENPP1, and less commonly ABCC6 mutations. Bisphosphonate treatment has been described to improve survival in ENPP1-positive GACI patients, but few studies have described bisphosphonate treatment in ABCC6-positive patients. Without therapy, patients will die before 6 months of age. Our patient is now 3 years old, former recipient twin of twin-to-twin transfusion syndrome (TTTS). Initial fetal echocardiogram at 19 weeks showed calcifications of the ascending aorta and pulmonary artery (PA). She underwent utero laser therapy, and despite resolution of the TTTS, her follow-up scans showed progressive calcification of the aorta and PA. Postnatal echocardiogram showed calcification and supravalvar stenosis of the aorta and PA. CT on day of life 6 showed calcifications in the PAs, aortic arch, and descending aorta. Quantification of valvular calcification can be difficult; in our patient, increasing outflow tract gradient on echocardiogram was used to monitor disease progression. Molecular testing revealed an ABCC6 gene mutation. She was started on weekly IV pamidronate (0.1–0.3 mg/kg/week) on day 8 of life then transitioned to oral etidronate (15–20 mg/kg/day). Given progressive supravalvar aortic and pulmonary stenosis, she underwent surgical repair with patch augmentation of the PA and ascending aorta at 4 months old. She has done well post-operatively, continuing on enteral bisphosphonate therapy with no side effects to date. Her identical twin was confirmed to have the same mutation and remains asymptomatic with no calcifications. Aggressive bisphosphonate therapy should be started as soon as possible in patients with infantile arterial calcinosis due to ABCC6 or ENPP1 mutations. Echocardiographic evaluation can be used to monitor disease progression by arterial gradients. Molecular testing is also essential to evaluate for possible co-morbidities in these patients and pregnancy management for the future.

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) and generalized arterial calcification of infancy (GACI) occur secondary to biallelic ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1) loss-of-function pathogenic variants. GACI is a life-threatening condition, often presenting in the neonatal period with heart failure and hypertension, caused by calcification of the media in large- and medium-sized arteries. ARHR2 typically manifests later in life. Children with ARHR2 commonly exhibit short stature, rachitic skeletal changes, progressive deformities of the lower limbs, skeletal fragility and bone/muscle pain. We present six cases of homozygous pathogenic variants in the ENPP1 gene causing ARHR2 and/or GACI. Case 1: Presented with lower limb deformities and pain with radiological evidence of rickets. Subsequent investigations displayed aortic and pulmonary arterial calcification. Case 2: Presented with lower limb deformities and knee pain. Confirmatory testing was undertaken following her brother’s (Case 1) diagnosis. Case 3: The diagnosis was made antenatally. Bisphosphonate treatment was instituted in both the pre- and post-natal periods due to the presence of extensive arterial calcifications. Rickets were noted by two years of age. Case 4: Presented with lower limb deformities and pain. There is neither any current evidence of arterial calcification nor hypertension. Case 5: Presented at 3 mo of age in cardiogenic shock with widespread calcification of large and medium-sized arteries. Bisphosphonate treatment was instituted. Case 6: Presented at 2 wk of age with right shoulder discomfort, with evidence of glenohumeral joint calcification. Further imaging revealed aortic, mediastinal, sternoclavicular joint and vertebral spinous process calcification. Case 1 and 2 were also found to have a heterozygous pathogenic ALPL variant consistent with hypophosphatasia. Clinical features, biochemistry, imaging and genetic analyses assist in the diagnosis of ARHR2 and GACI. Conventional therapy, oral phosphate and calcitriol for ARHR2 and bisphosphonates for GACI, have been utilized for many years. ENPP1 replacement treatment remains an exciting prospect for future management of ARHR2 and GACI secondary to loss of function of ENPP1.