ENPP1 enzyme replacement therapy improves ectopic calcification but does not rescue skeletal phenotype in a mouse model for craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a rare genetic bone disorder, characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. Craniofacial hyperostosis leads to the obstruction of neural foramina and neurological symptoms such as facial palsy, blindness, deafness, or severe headache. Mutations in ANKH (mouse ortholog ANK), a transporter of small molecules such as citrate and ATP, are responsible for autosomal dominant CMD. Knock-in (KI) mice carrying an ANKF377del mutation (AnkKI/KI ) replicate many features of human CMD. Pyrophosphate (PPi) levels in plasma are significantly reduced in AnkKI/KI mice. PPi is a potent inhibitor of mineralization. To examine the extent to which restoration of circulating PPi levels may prevent the development of a CMD-like phenotype, we treated AnkKI/KI mice with the recombinant human ENPP1-Fc protein IMA2a. ENPP1 hydrolyzes ATP into AMP and PPi. Male and female Ank+/+ and AnkKI/KI mice (n ≥ 6/group) were subcutaneously injected with IMA2a or vehicle weekly for 12 wk, starting at the age of 1 wk. Plasma ENPP1 activity significantly increased in AnkKI/KI mice injected with IMA2a (Vehicle/IMA2a: 28.15 ± 1.65/482.7 ± 331.2 mOD/min; p <.01), which resulted in the successful restoration of plasma PPi levels (Ank+/+ /AnkKI/KI vehicle treatment/AnkKI/KI IMA2a: 0.94 ± 0.5/0.43 ± 0.2/1.29 ± 0.8 µM; p <.01). We examined the skeletal phenotype by X-Ray imaging and µCT. IMA2a treatment of AnkKI/KI mice did not significantly correct CMD features such as the abnormal shape of femurs, increased bone mass of mandibles, hyperostotic craniofacial bones, or the narrowed foramen magnum. However, µCT imaging showed ectopic calcification near basioccipital bones at the level of the foramen magnum and on joints of AnkKI/KI mice. Interestingly, IMA2a treatment significantly reduced the volume of calcified nodules at both sites. Our data demonstrate that IMA2a is sufficient to restore plasma PPi levels and reduce ectopic calcification but fails to rescue skeletal abnormalities in AnkKI/KI mice under our treatment conditions.

Healthcare resource use associated with tumor-induced osteomalacia: a literature review.

CONTEXT: Tumor-induced osteomalacia (TIO) is an ultra-rare, paraneoplastic syndrome caused by tumors that secrete fibroblast growth factor 23 (FGF23). Initial signs and musculoskeletal symptoms can be non-specific and unrecognized, leading to long delays in diagnosis and treatment, which results in severe and progressive disability in patients with TIO. This review aimed to identify published evidence on healthcare resource use in TIO to better understand the burden of the disease. EVIDENCE ACQUISITION: A targeted literature review was conducted to identify publications reporting on disease characteristics and healthcare resource use associated with TIO. EVIDENCE SYNTHESIS: In total, 414 publications were included in the review, of which 376 were case reports. From the case reports, data on 621 patients were extracted. These patients had a mean (standard deviation) age of 46.3 (15.8) years; 57.6% were male. Mean time from first symptoms to diagnosis of TIO was 4.6 (4.7) years and, in cases where imaging tests were reported, patients underwent a mean of 4.1 (2.7) procedures. Tumor resection was attempted in 81.0% of patients and successful in 67.0%. Fracture was reported in 49.3% of patients. Results from association analyses demonstrated that longer time to diagnosis was associated with poorer tumor resection outcomes and a higher probability of tumor recurrence. Unfavorable tumor resection outcomes were associated with greater use of pharmacologic treatment and a greater likelihood of orthopedic surgery. CONCLUSION: TIO is associated with a substantial healthcare resource burden. Improvements in the diagnostic process could lead to better management of TIO, thereby benefiting patients and reducing that burden.

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.

Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract.

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

Hypophosphatemic rickets: An unexplained early feature of craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is an infrequently occurring skeletal dysplasia often caused by a mutation in ANKH. The most common features are early and progressive hyperostosis of craniofacial bones, which may cause obstruction of cranial nerves, and metaphyseal flaring of long bones. Rarely, rickets has been associated with CMD, occurring early in the course of the disease. We report an infant with CMD who presented with elevated serum alkaline phosphatase activity and low serum phosphorus at age 1 month and radiographic changes of rickets at 3 months of age. Further biochemical investigations revealed a high tubular reabsorption of phosphate and suppressed FGF23 level congruent with a deficit of phosphorus availability. Therapy with phosphorus was started at 4 months of age; calcitriol was subsequently added upon emergence of secondary hyperparathyroidism. A heterozygous pathogenic variant in ANKH c.1124_1126del (p.Ser375del) was identified. At 19 months of age therapy was discontinued in view of the corrected biochemical profile and radiographic improvement of rickets. ©The Authors. All rights reserved.

The efficacy and safety of burosumab in two patients with cutaneous skeletal hypophosphatemia syndrome.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is an ultra-rare mosaic disorder manifesting as skeletal dysplasia and FGF23-mediated hypophosphatemia, with some experiencing extra-osseous/extra-cutaneous manifestations, including both benign and malignant neoplasms. Like other disorders of FGF23-mediated hypophosphatemia including X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO), patients with CSHS have low serum phosphorus and active 1,25-dihydroxyvitamin D levels. Current treatment options for patients with CSHS include multiple daily doses of oral phosphorus and one or more daily doses of active vitamin D analog to correct the deficits. Recently, the fully human monoclonal antibody against FGF23 burosumab received US approval for the treatment of XLH and TIO, two rare diseases characterized by FGF23-mediated hypophosphatemia leading to rickets and osteomalacia. Given the similarities between the pathobiologies of these disorders and CSHS, we investigated the impact of burosumab on two patients, one pediatric and one adult, with CSHS who participated in separate, but similarly designed trials. In both the pediatric and adult patients, burosumab therapy was well-tolerated and contributed to clinically meaningful improvements in disease outcomes including normalization of phosphorus metabolism and markers of bone health, and improvements in skeletal abnormalities, fractures, and physical function. Reported adverse events were minimal, with only mild injection site reactions attributed to burosumab therapy. Together, these findings suggest that burosumab therapy is a promising therapeutic option for patients with CSHS.

Catalysis-Independent ENPP1 Protein Signaling Regulates Mammalian Bone Mass.

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1T238A mice. In contrast to Enpp1asj mice, which lack ENPP1, Enpp1T238A mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1asj mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear ß-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear ß-catenin signaling observed in Enpp1asj cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).

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.

Novel PHEX gene locus-specific database: Comprehensive characterization of vast number of variants associated with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemia, is caused by disrupting variants in the PHEX gene, located on the X chromosome. XLH is inherited in an X-linked pattern with complete penetrance observed for both males and females. Patients experience lifelong symptoms resulting from chronic hypophosphatemia, including impaired bone mineralization, skeletal deformities, growth retardation, and diminished quality of life. This chronic condition requires life-long management with disease-specific therapies, which can improve patient outcomes especially when initiated early in life. To centralize and disseminate PHEX variant information, we have established a new PHEX gene locus-specific database, PHEX LSDB. As of April 30, 2021, 870 unique PHEX variants, compiled from an older database of PHEX variants, a comprehensive literature search, a sponsored genetic testing program, and XLH clinical trials, are represented in the PHEX LSDB. This resource is publicly available on an interactive, searchable website (https://www.rarediseasegenes.com/), which includes a table of variants and associated data, graphical/tabular outputs of genotype-phenotype analyses, and an online submission form for reporting new PHEX variants. The database will be updated regularly with new variants submitted on the website, identified in the published literature, or shared from genetic testing programs.

Sustained Efficacy and Safety of Burosumab, a Monoclonal Antibody to FGF23, in Children With X-Linked Hypophosphatemia.

PURPOSE: In X-linked hypophosphatemia (XLH), excess fibroblast growth factor-23 causes hypophosphatemia and low calcitriol, leading to musculoskeletal disease with clinical consequences. XLH treatment options include conventional oral phosphate with active vitamin D, or monotherapy with burosumab, a monoclonal antibody approved to treat children and adults with XLH. We have previously reported outcomes up to 64 weeks, and here we report safety and efficacy follow-up results up to 160 weeks from an open-label, multicenter, randomized, dose-finding trial of burosumab for 5- to 12-year-old children with XLH. METHODS: After 1 week of conventional therapy washout, patients were randomized 1:1 to burosumab every 2 weeks (Q2W) or every 4 weeks (Q4W) for 64 weeks, with dosing titrated based on fasting serum phosphorus levels between baseline and week 16. From week 66 to week 160, all patients received Q2W burosumab. RESULTS: Twenty-six children were randomized initially into each Q2W and Q4W group and all completed treatment to week 160. In 41 children with open distal femoral and proximal tibial growth plates (from both treatment groups), total Rickets Severity Score significantly decreased by 0.9 ±â€…0.1 (least squares mean ±â€…SE; P < 0.0001) from baseline to week 160. Fasting serum phosphorus increases were sustained by burosumab therapy throughout the study, with an overall population mean (SD) of 3.35 (0.39) mg/dL, within the pediatric normal range (3.2-6.1 mg/dL) at week 160 (mean change from baseline P < 0.0001). Most adverse events were mild to moderate in severity. MAIN CONCLUSIONS: In children with XLH, burosumab administration for 160 weeks improved phosphate homeostasis and rickets and was well-tolerated. Long-term safety was consistent with the reported safety profile of burosumab. CLINICALTRIALS.GOV: NCT02163577.

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.

Burosumab for the Treatment of Tumor-Induced Osteomalacia.

Tumor-induced osteomalacia (TIO) is caused by phosphaturic mesenchymal tumors producing fibroblast growth factor 23 (FGF23) and is characterized by impaired phosphate metabolism, skeletal health, and quality of life. UX023T-CL201 is an ongoing, open-label, phase 2 study investigating the safety and efficacy of burosumab, a fully human monoclonal antibody that inhibits FGF23, in adults with TIO or cutaneous skeletal hypophosphatemia syndrome (CSHS). Key endpoints were changes in serum phosphorus and osteomalacia assessed by transiliac bone biopsies at week 48. This report focuses on 14 patients with TIO, excluding two diagnosed with X-linked hypophosphatemia post-enrollment and one with CSHS. Serum phosphorus increased from baseline (0.52 mmol/L) and was maintained after dose titration from week 22 (0.91 mmol/L) to week 144 (0.82 mmol/L, p < 0.0001). Most measures of osteomalacia were improved at week 48: osteoid volume/bone, osteoid thickness, and mineralization lag time decreased; osteoid surface/bone surface showed no change. Of 249 fractures/pseudofractures detected across 14 patients at baseline, 33% were fully healed and 13% were partially healed at week 144. Patients reported a reduction in pain and fatigue and an increase in physical health. Two patients discontinued: one to treat an adverse event (AE) of neoplasm progression and one failed to meet dosing criteria (receiving minimal burosumab). Sixteen serious AEs occurred in seven patients, and there was one death; all serious AEs were considered unrelated to treatment. Nine patients had 16 treatment-related AEs; all were mild to moderate in severity. In adults with TIO, burosumab exhibited an acceptable safety profile and was associated with improvements in phosphate metabolism and osteomalacia. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

Effects of Iron Isomaltoside vs Ferric Carboxymaltose on Hypophosphatemia in Iron-Deficiency Anemia: Two Randomized Clinical Trials.

Importance: Intravenous iron enables rapid correction of iron-deficiency anemia, but certain formulations induce fibroblast growth factor 23-mediated hypophosphatemia. Objective: To compare risks of hypophosphatemia and effects on biomarkers of mineral and bone homeostasis of intravenous iron isomaltoside (now known as ferric derisomaltose) vs ferric carboxymaltose. Design, Setting, and Participants: Between October 2017 and June 2018, 245 patients aged 18 years and older with iron-deficiency anemia (hemoglobin level ≤11 g/dL; serum ferritin level ≤100 ng/mL) and intolerance or unresponsiveness to 1 month or more of oral iron were recruited from 30 outpatient clinic sites in the United States into 2 identically designed, open-label, randomized clinical trials. Patients with reduced kidney function were excluded. Serum phosphate and 12 additional biomarkers of mineral and bone homeostasis were measured on days 0, 1, 7, 8, 14, 21, and 35. The date of final follow-up was June 19, 2018, for trial A and May 29, 2018, for trial B. Interventions: Intravenous administration of iron isomaltoside, 1000 mg, on day 0 or ferric carboxymaltose, 750 mg, infused on days 0 and 7. Main Outcomes and Measures: The primary end point was the incidence of hypophosphatemia (serum phosphate level <2.0 mg/dL) between baseline and day 35. Results: In trial A, 123 patients were randomized (mean [SD] age, 45.1 [11.0] years; 95.9% women), including 62 to iron isomaltoside and 61 to ferric carboxymaltose; 95.1% completed the trial. In trial B, 122 patients were randomized (mean [SD] age, 42.6 [12.2] years; 94.1% women), including 61 to iron isomaltoside and 61 to ferric carboxymaltose; 93.4% completed the trial. The incidence of hypophosphatemia was significantly lower following iron isomaltoside vs ferric carboxymaltose (trial A: 7.9% vs 75.0% [adjusted rate difference, -67.0% {95% CI, -77.4% to -51.5%}], P < .001; trial B: 8.1% vs 73.7% [adjusted rate difference, -65.8% {95% CI, -76.6% to -49.8%}], P < .001). Beyond hypophosphatemia and increased parathyroid hormone, the most common adverse drug reactions (No./total No.) were nausea (iron isomaltoside: 1/125; ferric carboxymaltose: 8/117) and headache (iron isomaltoside: 4/125; ferric carboxymaltose: 5/117). Conclusions and Relevance: In 2 randomized trials of patients with iron-deficiency anemia who were intolerant of or unresponsive to oral iron, iron isomaltoside (now called ferric derisomaltose), compared with ferric carboxymaltose, resulted in lower incidence of hypophosphatemia over 35 days. However, further research is needed to determine the clinical importance of this difference. Trial Registration: ClinicalTrials.gov Identifiers: NCT03238911 and NCT03237065.

Frequent overexpression of klotho in fusion-negative phosphaturic mesenchymal tumors with tumorigenic implications.

Phosphaturic mesenchymal tumors (PMT) are tumors that cause hypophosphatemia/osteomalacia chiefly by secreting FGF23. We have identified FN1-FGFR1/FGF1 fusion genes in nearly half of PMT, suggesting a central role of FGFR1 pathways in the pathogenesis of PMT. Tumorigenic drivers are unknown for tumors where previous study detected neither fusion, including many in bone, where FISH failed because of tissue decalcification. To identify alternative fusions in PMT without known fusions, as well as to validate the positive FISH results and characterize the fusion junctions, 34 PMT were studied, including 12 with known FN1-FGFR1 fusion by FISH (Group A), 2 with FN1-FGF1 (B), 12 with neither fusion (C), and 8 with previous acid-based decalcification and hence unknown fusion status (D). In total, 23 archival samples were subjected to anchored multiplex PCR-based RNA-sequencing (AMP-seq) with primers targeting FN1, genes encoding the FGF/FGFR families, and KL (α-Klotho); five Group C cases were also studied with whole-transcriptomic and exome-captured RNA sequencing, respectively. The AMP-seq results were consistent with previous FISH and/or transcriptomic sequencing data, except in one old Group A sample. One case had a novel FGFR1 exon 9 breakpoint, confirmed by genomic DNA sequencing. One Group D bone tumor was found to harbor FN1-FGF1. All 3 RNA-sequencing platforms failed to identify convincing fusion genes in Group C (N = 10), which instead expressed significantly higher levels of either KL or KLB. This result was further confirmed with KL and KLB RNA CISH semi-quantification (RNAscope). Our results demonstrated the utility of AMP-seq, which was compromised by decalcification and prolonged archiving. Of potential importance, fusion-negative PMT frequently overexpressed α-Klotho (or instead ß-Klotho less commonly), whose role as an obligatory co-receptor for FGF23-FGFR1 binding suggests its aberrant expression in osteocytes/osteoblasts might result in an FGF23-FGFR1 autocrine loop that in turn drives the overexpression of FGF23 and tumorigenesis through activated FGFR pathways.

Severity of reduced bone mineral density and risk of fractures in long-term survivors of childhood leukemia and lymphoma undergoing guideline-recommended surveillance for bone health.

BACKGROUND: Survivors of childhood leukemia/lymphoma are at increased risk for reduced bone mineral density (BMD). The authors sought to determine the frequency of reduced BMD detected by off-therapy surveillance, factors associated with reduced BMD, and the association of reduced BMD with fractures. METHODS: This cross-sectional study included childhood leukemia/lymphoma survivors attending 2 survivorship clinics who received guideline-recommended BMD surveillance ≥2 years post-therapy with dual-energy x-ray absorptiometry (from January 1, 2004 to August 31, 2016). Lumbar spine BMD z-scores were height-for-age-adjusted. Low and very low BMD were >1 SD and >2 SDs below norms, respectively. Treatment, chronic conditions, and fractures were abstracted from medical records. Logistic regression was used to examine the association of low BMD with patient/treatment factors and fractures. RESULTS: In total, 542 patients (51.5% female) with a mean age of 15.5 years (range, 4.4-52.2 years) who were 6 years post-therapy (range, 2.0-35.1 years) were evaluated, including 116 who reported post-therapy fractures. Lumbar spine low BMD was identified in 17.2% of survivors, and very low BMD was identified in 3.5% of survivors, but frequencies varied considerably between subgroups; 10.8% of survivors aged 15 to 19 years at diagnosis had very low BMD. In multivariable analyses, older age at diagnosis, white race, and being underweight were significantly associated with low BMD. Survivors with low BMD had greater odds of nondigit fractures (odds ratio, 2.2; 95% CI, 1.3-3.7) and specifically long-bone fractures (odds ratio, 2.7; 95% CI, 1.5-4.7). CONCLUSIONS: In this study of childhood leukemia/lymphoma survivors undergoing guideline-recommended dual-energy x-ray absorptiometry surveillance, patients who were older at diagnosis, white, and underweight were at the highest risk for lumbar spine low BMD. Low BMD was associated with a greater risk of fractures, emphasizing the clinical importance of surveillance.

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.

High dose vitamin D supplementation does not rescue bone loss following Roux-en-Y gastric bypass in female rats.

Postoperative bone loss and increased fracture risk associated with Roux-en-Y gastric bypass (RYGB) have been attributed to vitamin D/calcium malabsorption and resultant secondary hyperparathyroidism (HPT). Adequate vitamin D supplementation (VDS), particularly in an older female population, reduces incidence of secondary HPT but the effect on bone loss and fracture risk remains unclear. To investigate whether VDS corrects the RYGB bone phenotype, 41 obese adult female rats were randomized to RYGB with 1000 IU (R1000) or 5000 IU (R5000) vitamin D/kg food or a sham surgical procedure with either paired (PF) or ad libitum (AL) feeding. Bone turnover markers, urinary calcium/creatinine ratio (CCR), and serum calciotropic and gut hormones were assessed throughout a 14-week postoperative period. Femurs were analyzed by micro-computed tomography (µCT), three-point bending test, and histomorphometry. 1000 IU animals had low 25­hydroxyvitamin D (25(OH)D), high serum parathyroid hormone (PTH), and very low urine CCR levels. 5000 IU corrected the 25(OH)D and secondary HPT but did not increase urine CCR or serum levels of 1,25­dihydroxyvitamin D (1,25(OH)D) significantly between RYGB groups. Compared to sham animals at 14 weeks, RYGB animals had significantly higher serum osteocalcin (OCN) and C-terminal telopeptide (CTX) levels. The gut hormone peptide tyrosine tyrosine hormone (PYY) was higher in the RYGB groups, and leptin was lower. µCT and biomechanical testing revealed RYGB females had decreased cortical and trabecular bone volume and weaker, stiffer bone than controls. Histomorphometry showed decreased bone volume and increased osteoid volume with increased mineral apposition rate in RYGB compared to controls. No differences in bone phenotype were identified between 1000 IU and 5000 IU groups, and osteoclast numbers were comparable across all four groups. Thus, in our model, 5000 IU VDS corrected vitamin D deficiency and secondary HPT but did not rescue RYGB mineralization rate nor the osteomalacia phenotype. Longer studies in this model are required to evaluate durability of these detrimental effects. Our findings not only underscore the importance of lifelong repletion of both calcium and vitamin D but also suggest that additional factors affect skeletal health in this population.

Continued Beneficial Effects of Burosumab in Adults with X-Linked Hypophosphatemia: Results from a 24-Week Treatment Continuation Period After a 24-Week Double-Blind Placebo-Controlled Period.

Burosumab, a fully human monoclonal antibody to FGF23, is the only approved treatment for X-linked hypophosphatemia (XLH), a rare genetic disorder characterized by renal phosphate wasting and substantial cumulative musculoskeletal morbidity. During an initial 24-week randomized, controlled trial, 134 adults with XLH received burosumab 1 mg/kg (n = 68) or placebo (n = 66) every 4 weeks. After 24 weeks, all subjects received open-label burosumab until week 48. This report describes the efficacy and safety of burosumab during the open-label treatment period. From weeks 24-48, serum phosphorus concentrations remained normal in 83.8% of participants who received burosumab throughout and were normalized in 89.4% who received burosumab after placebo. By week 48, 63.1% of baseline fractures/pseudofractures healed fully with burosumab, compared with 35.2% with burosumab after placebo. In both groups, burosumab was associated with clinically significant and sustained improvement from baseline to week 48 in scores for patient-reported outcomes of stiffness, pain, physical function, and total distance walked in 6 min. Rates of adverse events were similar for burosumab and placebo. There were no fatal adverse events or treatment-related serious adverse events. Nephrocalcinosis scores did not change from baseline by more than one grade at either week 24 or 48. These data demonstrate that in participants with XLH, continued treatment with burosumab is well tolerated and leads to sustained correction of serum phosphorus levels, continued healing of fractures and pseudofractures, and sustained improvement in key musculoskeletal impairments.