Effects of food, fasting, and exercise on plasma pyrophosphate levels and ENPP1 activity in healthy adults.

BACKGROUND: Inorganic pyrophosphate (PPi) is highly regulated as it plays a critical role in the regulation of physiological mineralization. Dysregulation of plasma PPi is associated with skeletal hypomineralization and pathogenic mineralization in soft connective tissue, arteries, and heart valves. There is no standard approach to measuring PPi, making it difficult to establish PPi as a biomarker of mineralization disorders. This study aims to determine the impact of time of day, meals, or exercise on plasma PPi homeostasis using a highly sensitive PPi assay. METHODS: In this single-center trial, a clinical laboratory improvement amendment (CLIA) validated modified sulfurylase-based adenosine 5-triphosphate (ATP) assay was used to measure PPi levels throughout the day in 10 healthy adults under 3 conditions; normal diet (non-fasting), fasting, and normal diet with exercise. Serum ectonucleotide pyrophosphatase/phosphodiesterase 1 activity (ENPP1; an enzyme that produces PPi) was also measured to determine whether these conditions influence PPi levels through ENPP1 activity. RESULTS: There is a circadian increase in mean PPi levels under fasting and non-fasting conditions between 8 am and 6 pm, followed by a rapid return to baseline overnight. A circadian increase in ENPP1 activity was also measured under fasting but was lost under non-fasting conditions. Meals increased the individual variability of PPi levels when compared to the same individual fasting. PPi levels and ENPP1 activity exhibited a short-term increase after intense exercise. We found PPi ranges from 1465 nM to 2969 nM (mean 2164 nM) after fasting overnight. Within this range, there was lower intra-subject variability in PPi, suggesting that each individual has a uniquely regulated normal PPi range. CONCLUSION: Plasma levels of PPi can be reliably measured after an overnight fast and show promise as a biomarker of mineralization disorders.

ENPP1 deficiency: A clinical update on the relevance of individual variants using a locus-specific patient database.

Loss-of-function variants in the ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) cause ENPP1 Deficiency, a rare disorder characterized by pathological calcification, neointimal proliferation, and impaired bone mineralization. The consequence of ENPP1 Deficiency is a broad range of age dependent symptoms and morbidities including cardiovascular complications and 50% mortality in infants, autosomal recessive hypophosphatemic rickets type 2 (ARHR2) in children, and joint pain, osteomalacia and enthesopathies in adults. Recent research continues to add to the growing clinical presentation profile as well as expanding the role of ENPP1 itself. Here we review the current knowledge on the spectrum of clinical and genetic findings of ENPP1 Deficiency reported in patients diagnosed with GACI or ARHR2 phenotypes using a comprehensive database of known ENPP1 variants with associated clinical data. A total of 108 genotypes were identified from 154 patients. Of the 109 ENPP1 variants reviewed, 72.5% were demonstrably disease-causing, a threefold increase in pathogenic/likely pathogenic variants over other databases. There is substantial heterogeneity in disease severity, even among patients with the same variant. The approach to creating a continuously curated database of ENPP1 variants accessible to clinicians is necessary to increase the diagnostic yield of clinical genetic testing and accelerate diagnosis of ENPP1 Deficiency.

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

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

Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency leads to cardiovascular calcification in infancy, fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets in childhood, and osteomalacia in adulthood. Excessive enthesis mineralization and cervical spine fusion have been previously reported in patients with biallelic ENPP1 deficiency, but their effect on quality of life is unknown. We describe additional musculoskeletal complications in patients with ENPP1 deficiency, namely osteoarthritis and interosseous membrane ossification, and for the first time evaluate health-related quality of life (HRQoL) in patients with this disease, both subjectively via narrative report, and objectively via the Brief Pain Inventory-Short Form, and a Patient Reported Outcome Measurement Information System Physical Function (PROMIS PF) short form. Residual pain, similar in magnitude to that identified in adult patients with X-linked hypophosphatemia, was experienced by the majority of patients despite use of analgesic medications. Impairment in physical function varied from mild to severe. To assess murine ENPP1 deficiency for the presence of enthesopathy, and for the potential response to enzyme replacement therapy, we maintained Enpp1asj/asj mice on regular chow for 23 weeks and treated cohorts with either vehicle or a long-acting form of recombinant ENPP1. Enpp1asj/asj mice treated with vehicle exhibited robust calcification throughout their Achilles tendons, whereas two-thirds of those treated with ENPP1 enzyme replacement exhibited complete or partial suppression of the Achilles tendon calcification. Our combined results document that musculoskeletal complications are a significant source of morbidity in biallelic ENPP1 deficiency, a phenotype which is closely recapitulated in Enpp1asj/asj mice. Finally, we show that a long-acting form of recombinant ENPP1 prevents the development of enthesis calcification at the relatively modest dose of 0.3 mg/kg per week, suggesting that suppression of enthesopathy may be attainable upon dose escalation. © 2021 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.

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

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

Eculizumab in paroxysmal nocturnal hemoglobinuria with Budd-Chiari syndrome progressing despite anticoagulation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive, life-threatening disorder characterized by chronic intravascular hemolysis caused by uncontrolled complement activation. Hepatic vein thrombosis (Budd-Chiari syndrome) is common in PNH patients. This case report describes the response to eculizumab (a humanized monoclonal antibody that inhibits terminal complement activation) in a 25-year-old male with progressive liver function deterioration despite standard anticoagulation therapy and transjugular intrahepatic porto-systemic shunt. The patient presented with anemia, severe thrombocytopenia, headache, abdominal pain, and distention. He was diagnosed with PNH, cerebral vein thrombosis, and Budd-Chiari syndrome. Despite adequate anticoagulation, diuretic administration, and placement of a transjugular shunt, additional thrombotic events and progressive liver damage were observed. Eculizumab therapy was initiated, resulting in rapid blockade of intravascular hemolysis, increased platelet counts, ascites resolution, and liver function recovery, all of which are presently sustained. Since starting eculizumab the patient has had no further thrombotic events and his quality of life has dramatically improved. This is the first report to confirm the role of complement-mediated injury in the progression of Budd-Chiari syndrome in a patient with PNH. This case shows that terminal complement blockade with eculizumab can reverse progressive thromboses and hepatic failure that is unresponsive to anticoagulation therapy and suggests that early initiation of eculizumab should be included in the therapeutic regimen of patients with PNH-related Budd-Chiari syndrome.