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.

Bisphosphonate therapy in an infant with generalized arterial calcification with an ABCC6 mutation.

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.

Inherited Arterial Calcification Syndromes: Etiologies and Treatment Concepts.

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

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

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

Generalized Arterial Calcification of Infancy (GACI).

Generalized arterial calcification of infancy (GACI) is an ultra-rare autosomal recessive disorder associated with pathogenic variants in ENPP1, the major gene involved in this condition, and in ABCC6, which is involved in a small fraction of affected individuals. Loss-of-function pathogenic variants of ENPP1 and ABCC6 lead to perturbations in the PPi/Pi ratio, thereby promoting hydroxyapatite mineralization in peripheral tissues. GACI is initially characterized by an abnormal ectopic mineralization process in arteries and soft tissue. Nearly half of the patients die within the first 6 months of life from cardiovascular complications, hence the poor prognosis associated with an early diagnosis. In recent years, progress has been made in our understanding of the long-term natural history of GACI, the intricate symptoms due to vascular calcifications, the overmineralization of soft tissues, of hypophosphatemia designated as ARHR2, and of the consequences such as undermineralization of the skeleton, but also of the features possibly seen in pseudoxanthoma elasticum (PXE). Indeed, GACI, PXE, and ARHR2 share common pathophysiological pathways and clinical features beyond the vascular calcifications. Treatment options for severe forms of GACI are mostly based on symptomatic management, including the option of starting bisphosphonates early after birth, such as etidronate and pamidronate, analogues of PPi. Follow-up within an expert and coordinated multidisciplinary team includes treatment of arterial hypertension, calcitriol and phosphorus adjustments, hearing aids, and early detection of possible angioid streaks. It is hoped that ongoing basic and clinical research will lead to the development of effective therapies that specifically target the abnormal PPi regulation and the other mechanisms involved in this disorder.

The Purinergic Nature of Pseudoxanthoma Elasticum.

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.

Outcomes following gel-based autologous chondrocyte implantation for articular cartilage defects of the knee.

BACKGROUND: Gel-based autologous chondrocyte implantation (GACI) enables a simpler and more effective delivery of chondrocytes with reproducible three-dimensional structural restoration of the articular cartilage surface. There is limited documentation of medium-term outcomes. This study assessed safety and effectiveness of GACI for treatment of cartilage defects of the knee. METHODS: This multicentric retrospective study was conducted across eight hospitals in India. Patients who had undergone GACI (CARTIGROW®) between 2008 and 2014 for the treatment of focal articular cartilage defects of the knee (mean defect size 4.5 ± 5.8 cm2) in limbs with normal alignment were analyzed. Primary outcomes were changes in Lysholm Knee Scoring Scale score, and Knee Outcome Sports Activity Scale (SAS). RESULTS: A total of 107 patients (110 knee joints) with mean age 31.0 ± 10.5 years were included. The mean follow-up was 9.8 ± 1.5 years (range 7.85-13.43). Majority had osteochondritis dissecans (n = 51; 46.4%). The mean Lysholm Knee Scoring Scale score (81.23 ± 13.21 vs. 51.32 ± 17.89; p < 0.0001) and SAS score (80.93 ± 8.26 vs. 28.11 ± 12.28; p < 0.0001) improved significantly at follow-up as compared to pre-operative. Magnetic Resonance Observation of Cartilage Repair Tissue score in 39 patients at minimum 2 years follow-up was 84.5 ± 4.3. Among 30 patients who were playing sports before treatment, 17 patients (56.7%) could return to the same or higher level of sports post-transplantation. No major intra-operative or post-operative complications were noted. Four patients warranted revision surgery. CONCLUSION: GACI is an effective treatment option for large focal articular cartilage defects of the knee with a low complication rate and revision rate and significant improvement in functional scores.

Quantitative correlation of ENPP1 pathogenic variants with disease phenotype.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.

Autosomal recessive hypophosphatemic rickets type 2 due to ENPP1 deficiency (ARHR2).

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2; MIM #613312) is a very rare disorder caused by biallelic loss-of-function mutations in the ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene. ENPP1 deficiency encompasses a spectrum of phenotypes that includes, in addition to ARHR2, generalized arterial calcification of infancy (GACI), ossification of the posterior longitudinal ligament (OPLL), and pseudoxanthoma elasticum. ARHR2 can be found in GACI survivors, but it may also be the first manifestation of ENPP1 deficiency. Although the precise mechanisms are not fully elucidated, patients with GACI and ARHR2 have elevated serum FGF23 levels, leading to renal phosphate wasting and hypophosphatemia. As a result, the clinical and radiological phenotype of ARHR2 patients is very similar to that of patients affected with other forms of hypophosphatemic rickets, such as X-linked hypophosphatemia. Patients show signs of rickets (abnormal mineralization of growth plates in children) and osteomalacia (abnormal bone mineralization in children and adults) of varying severity. Clinical manifestations specific to ENPP1 loss-of-function mutations and common to GACI, such as ectopic calcifications (valvular, arterial, or periarticular), deafness, OPLL, and PXE, may also be found. Genetic confirmation of the disease is important so as to ensure that patients receive the appropriate treatment or have the opportunity to participate in clinical trials to evaluate the safety and efficacy of novel and promising recombinant enzyme therapies.

ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

Gel-Based Autologous Chondrocyte Implantation (GACI) in the Chondral Defects of the Knee: An Observational Study.

Introduction: Gel-based autologous chondrocyte implantation (GACI) is known to have superior results when compared to conventional autologous chondrocyte implantation (ACI) in terms of delivery of chondrocytes to the articular cartilage surface with reproducible three-dimensional structural restoration. This study aims to evaluate the short-term outcomes of gel-based autologous chondrocyte implantation (GACI) for the treatment of large focal articular cartilage defects of the knee. Methods: This was a prospective observational study among 25 patients who underwent GACI. Primary outcome measures included Lysholm Knee Scoring Scale and IKDC score and secondary outcome measures included MRI assessment of cartilage repair using MOCART. Results: Mean age of the population was 39.8 ± 7.5 years. The study found a highly significant improvement in both Lysholm knee score (pre-op: 45.1 to post-op: 72.4) and IKDC score (pre-op: 36.7 to post-op: 78.5) (p < 0.001) at the final follow-up of 24 months, even with the mean defect size being 4.5 ± 5.8 cm2. Postoperative MRI showed a mean MOCART score improvement from 39.4 to 67.4 at the final follow-up. No major complications were observed. Conclusion: GACI is an effective and safe treatment option for large focal articular cartilage defects around the knee, with significant improvement in functional scores and low revision rates at medium-term follow-up.

Severe early-onset manifestations of generalized arterial calcification of infancy (mimicking severe coarctation of the aorta) with ABCC6 gene variant - Case report and literature review.

Introduction: Generalized arterial calcification of infancy (GACI) is a rare cause of infantile heart failure and systemic hypertension with a poor prognosis, characterized by extensive calcification and proliferation of the intimal layer of large and medium sized arteries. Case report: We present the first case report of successful surgical treatment of severe aortic arch obstruction by calcified plaques mimicking severe coarctation of the aorta and the outcome (of bisphosphonate therapy) in a newborn with GACI. Furthermore, we report the identification of a variant in ATP Binding Cassette Subfamily C, Member 6 (ABCC6) gene, possibly associated with severe early-onset manifestations of GACI. Conclusion: This case report highlights the importance of considering GACI in an infant with heart failure, systemic hypertension, and evidence of increased echogenicity of the arterial vessels. We noted the favorable outcome in improving the aortic calcification in our patient after surgical treatment and bisphosphonates therapy. Early diagnosis and treatment improve the long-term prognosis. A better understanding of this rare genetic disease could lead to new therapeutic strategies.

Estimation of ENPP1 deficiency genetic prevalence using a comprehensive literature review and population databases.

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.

Serum biomarkers for arterial calcification in humans: A systematic review.

Aim: To clarify the role of mediators of ectopic mineralization as biomarkers for arterial calcifications. Methods: MEDLINE and Embase were searched for relevant literature, until January 4th 2022. The investigated biomarkers were: calcium, phosphate, parathyroid hormone, vitamin D, pyrophosphate, osteoprotegerin, receptor activator of nuclear factor-kappa B ligand (RANKL), fibroblast growth factor-23 (FGF-23), Klotho, osteopontin, osteocalcin, Matrix Gla protein (MGP) and its inactive forms and vitamin K. Studies solely performed in patients with kidney insufficiency or diabetes mellitus were excluded. Results: After screening of 8985 articles, a total of 129 articles were included in this systematic review. For all biomarkers included in this review, the results were variable and more than half of the studies for each specific biomarker had a non-significant result. Also, the overall quality of the included studies was low, partly as a result of the mostly cross-sectional study designs. The largest body of evidence is available for phosphate, osteopontin and FGF-23, as a little over half of the studies showed a significant, positive association. Firm statements for these biomarkers cannot be drawn, as the number of studies was limited and hampered by residual confounding or had non-significant results. The associations of the other mediators of ectopic mineralization with arterial calcifications were not clear. Conclusion: Associations between biomarkers of ectopic mineralization and arterial calcification are variable in the published literature. Future longitudinal studies differentiating medial and intimal calcification could add to the knowledge of biomarkers and mechanisms of arterial calcifications.

Generalized Arterial Calcification of Infancy Type 1 (GACI1): Identification of a Novel Pathogenic Variant (c.1715T>C (p.Leu572Ser)).

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.

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) due to ENPP1-deficiency.

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.

Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization.

Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.

Skeletal abnormalities secondary to antenatal etidronate treatment for suspected generalised arterial calcification of infancy.

BACKGROUND: Generalised arterial calcification of infancy (GACI) is a rare disorder characterised by the deposition of hydroxyapatite crystals within the vessel walls. It is associated with a high mortality rate. Bisphosphonates have been used with some success in the treatment of GACI. However, there is a paucity of data on the antenatal use of bisphosphonates for GACI. In this paper, we report development of the skeletal changes suggestive of hypophosphatasia (HPP) in an infant with GACI, whose mother was treated with etidronate during pregnancy. CASE REPORT: A Caucasian infant boy had a suspected antenatal diagnosis of GACI based on the findings suggestive of calcification of the annulus of the tricuspid valve and wall of the right ventricular (RV) outflow tract and main pulmonary artery on foetal echocardiography and the genetic analysis which showed a pathogenic heterozygous mutation in ABCC6. Based on these findings, mother was started on etidronate treatment from 26 weeks of gestation. A healthy male baby was delivered at 38 weeks of gestation. Initial postnatal echocardiogram on day 1 of life was normal with good biventricular function; subtle changes suggestive of microcalcifications were detected on the CT angiography. Serum calcium, phosphate, alkaline phosphatase and renal profile were normal. Further, the serum inorganic pyrophosphate (PPi) level was significantly low. Skeletal changes suggestive of HPP were seen on the radiographs. The baby developed cardiac dysfunction on day 4 of life with evidence of ischaemic changes on electrocardiogram (ECG).Treatment with etidronate was started in view of probable evolving coronary calcifications. Despite treatment with cardiac supportive measures and bisphosphonate, he succumbed to death in the third week of life. DISCUSSION: We believe, this is the first report of skeletal changes suggestive of HPP, arising secondary to antenatal etidronate (first generation bisphosphonate) used for the treatment of suspected GACI due to a heterozygous ABCC6 mutation.

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency.

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 < -2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Hyperphosphatemia with low FGF7 and normal FGF23 and sFRP4 levels in the circulation characterizes pediatric hypophosphatasia.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP in healthy individuals is on cell surfaces richly in bone, liver, and kidney. Thus, TNSALP natural substrates accumulate extracellularly in HPP, including inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation and growth. Superabundance of extracellular PPi (ePPi) in HPP impairs mineralization of bones and teeth, often leading to rickets during childhood and osteomalacia in adult life and to tooth loss at any age. HPP's remarkably broad-ranging severity is largely explained by nearly four hundred typically missense mutations throughout the ALPL gene that are transmitted as an autosomal dominant or autosomal recessive trait. In the clinical laboratory, the biochemical hallmark of HPP is low serum ALP activity (hypophosphatasemia). However, our experience indicates that hyperphosphatemia from increased renal reclamation of filtered inorganic phosphate (Pi) is also common. Herein, from our prospective single-center study, we document throughout the clinical spectrum of non-lethal pediatric HPP that hyperphosphatemia reflects increased renal tubular threshold maximum for phosphorus adjusted for the glomerular filtration rate (TmP/GFR). To explore its pathogenesis, we studied mineral metabolism and quantitated circulating levels of three phosphatonins [fibroblast growth factor 23 (FGF23), secreted frizzled-related protein 4 (sFRP4), and fibroblast growth factor 7 (FGF7)] in 41 pediatric patients with HPP, 73 with X-linked hypophosphatemia (XLH), and 15 healthy pediatric control (CTR) subjects. The HPP and XLH cohorts had normal serum total and ionized calcium and parathyroid hormone levels (Ps > 0.10) and uncompromised glomerular filtration. In XLH, serum FGF23 was characteristically elevated (P < 0.0001) and despite hypophosphatemia sFRP4 was normal (P > 0.4) while FGF7 was low (P < 0.0001). In HPP, despite hyperphosphatemia serum FGF23 and sFRP4 were normal (Ps > 0.8) while FGF7 was low (P < 0.0001). Subsequently, in rats, we confirmed that FGF7 is phosphaturic. Thus, hyperphosphatemia in non-lethal pediatric HPP is associated with phosphatonin insufficiency together with, as we discuss, ePPi excess and diminished renal TNSALP activity.