Hypophosphatasia (HPP) is a rare metabolic bone disorder that can present with a wide spectrum of skeletal and extraskeletal signs and symptoms. Biochemically, HPP is characterized by a decreased activity of the tissue nonspecific alkaline phosphatase (TNSALP) and subsequent accumulation of inorganic pyrophosphate (PPi). Fractures in patients with HPP can show delayed healing or even progress to nonunion. In pediatric patients, fractures can be particularly debilitating, as children usually engage in high levels of physical activity. The 16-year-old girl, carrying a pathogenic variant in the ALPL gene, presented 17weeks (4months) after the initial diagnosis of a fracture at the base of the fifth metatarsal (MT-V). The patient was still symptomatic, and magnetic resonance imaging and cone beam computed tomography (CBCT) showed no radiological signs of healing despite immobilization. Given the prolonged absence of fracture consolidation, TNSALP enzyme replacement therapy with asfotase alfa (AA) was initiated and dosed according to body weight, in line with approved pediatric regimens. Thirteen weeks after initiation of AA, CBCT demonstrated full radiological consolidation. Furthermore, bone mineral density (BMD) in the fracture gap increased by 36%, returning to the average BMD level of the MT-V. Here we present, to the best of our knowledge, the first pediatric case in which treatment with AA supported healing of a delayed union stress fracture in the context of clinically diagnosed and genetically supported HPP. Our findings support considering AA initiation in pediatric patients diagnosed with HPP who present delayed fracture healing despite standard conservative management.

AVP1-mediated pyrophosphate homeostasis coordinates calcium-dependent cellulose synthesis and autoimmunity during leaf growth.

Cobalt-ferrocene MOF-based colorimetric biosensing platform for naked-eye detection of human papillomavirus.

Inorganic pyrophosphatase-1 (PPa1) is an essential enzyme proposed to limit accumulation of the ubiquitous metabolic byproduct inorganic pyrophosphate, yet its role beyond a general housekeeping function remains poorly understood. We generated viable hypomorphic alleles of PPa1 through random mutagenesis in mice and unexpectedly found that PPa1 insufficiency causes a lysosomal storage disease of the bone marrow. Mutant mice developed impaired hematopoiesis and defective skeletal mineralization in a hematopoietic-intrinsic manner. The bone marrow was infiltrated by glycolipid-laden macrophages that were marked by high Spp1 and CD14 expression, blunted lysosomal acidification, metabolic stress, and a distinct inflammatory transcriptional program. Consistent with a lysosomal storage defect, PPa1-deficient bone marrow accumulated elevated levels of long-chain glucosyl-sphingolipids and sphingosine. These findings identify PPa1 as a previously unrecognized regulator of lysosomal function in myeloid cells that restrains inflammatory macrophage expansion and prevents bone marrow lysosomal storage pathology.

RNA synthesis by eukaryotic polymerases requires existing polynucleotides to serve as templates or primers. Here, we describe primer- and template-free RNA generation by human terminal nucleotidyltransferase 4B (TENT4B) via de novo polymerization of free nucleotides. We observed that recombinant TENT4B (rTENT4B) consumes ATP to yield inorganic pyrophosphate in the absence of a primer or template, concurrent with the appearance of oligomeric poly-adenosine RNA products. Remarkably, 5' labels on γ-phosphate-modified ATP or GTP are retained during polymerization in the presence of unlabeled nucleotide triphosphates (NTPs). These polymers are created at a similar efficiency irrespective of the inclusion of a primer, indicating robust RNA synthesis by rTENT4B from free NTPs. While canonical purine NTPs are favored, nucleotide diphosphates can also serve as substrates for rTENT4B-mediated de novo RNA polymerization. rTENT4B-mediated RNA synthesis using free adenosine nucleotides shows high processivity to generate 1000s-mers, whereas guanosine nucleotide polymerization is strongly and uniformly self-limited and yields a 3'-exonuclease-resistant oligonucleotide. Interrogation of other RNA polymerases reveals potential capacity for de novo polymerization using free ATP, albeit at significantly higher substrate concentrations and lower efficiency compared to rTENT4B. Our data provide definitive evidence of efficient template-free de novo RNA synthesis by a eukaryotic polymerase.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a newly discovered histidine phosphatase that can remove histidine-linked phosphate groups from proteins, but its function is unclear. Given that the LHPP protein might participate in histidine phosphorylation, it is receiving increasing attention from researchers. Recent evidence has suggested that LHPP is a novel tumor suppressor with a crucial role in cancers, as its expression is decreased in different cancers. Previous studies have confirmed that abnormal LHPP levels can lead to overactivation of pathways such as the PI3K/AKT pathway during the course of tumor pathogenesis. In addition, LHPP has been confirmed as a susceptibility gene for depression, which has triggered many related studies. However, the current understanding of the functions of LHPP in diseases is relatively limited, and a systematic summary of its mechanism is lacking. Therefore, this paper reviews relevant studies of LHPP to clarify the mechanism of its involvement in cancers and nervous system diseases and provides new strategies for disease research involving LHPP.

Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2) caused by biallelic ENPP1 mutations is a rare disorder with a broad phenotypic spectrum. We describe three affected siblings from a consanguineous family who presented with markedly heterogeneous clinical features. The proband exhibited classical signs of rickets with progressive lower-limb deformities, short stature, and elevated alkaline phosphatase. Her older sister demonstrated limited elbow extension, conductive hearing loss, and vascular stenoses, while the youngest sibling developed early biochemical abnormalities before overt skeletal manifestations of rickets emerged. All affected children had hypophosphatemia, reduced TmP/GFR, and elevated or inappropriately normal FGF23 concentrations, consistent with FGF23-mediated phosphate wasting. Notably, plasma inorganic pyrophosphate (PPi) levels were markedly reduced in the affected children and mildly reduced in the carriers of monoallelic mutation. Genetic testing identified a homozygous ENPP1 variant, c.2559_2561del p.(Leu854del), which was essential for establishing the diagnosis and distinguishing ARHR2 from other hereditary forms of hypophosphatemic rickets. The father had low lumbar spine bone mineral density. These cases highlight the clinical heterogeneity of ENPP1 deficiency and reinforce the essential role of genetic testing in establishing the correct diagnosis.

Biochemical characterization and identification of catalytic residues of the thermostable inorganic pyrophosphatase from Thermococcus litoralis.

Pathological reduction in enzymatic activity of the tissue-non-specific alkaline phosphatase (TNSALP) is the molecular hallmark of hypophosphatasia (HPP), a group of rare inborn systemic diseases, mainly characterized by pathological affections of calcified tissue mineralization and the musculoskeletal system. The disease, in all clinical forms, is biochemically characterized by variable degrees of chronically reduced activity of circulating total alkaline phosphatase (ALP). Repeated detection of low values of ALP activity is mandatory to diagnose the presence of HPP, but, alone, it is not sufficient for the diagnosis of the disease. Detection of increased circulating levels of one of the main natural substrates of TNSALP, the pyridoxal 5'-phosphate (PLP), is needed to biochemically confirm the diagnosis of HPP. Urinary and/or blood levels of phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi), two other natural substrates of TNSALP, can be elevated in a percentage of HPP patients. The contemporary biochemical evaluation of ALP activity and its target substrates is of great help in the diagnosis of HPP, and also for the monitoring of a patient's response to enzymatic replacement therapy or other pharmacological treatments. Here, we describe and discuss possibilities and challenges of biochemical screenings for HPP, based also on the experience gained in our analysis laboratory.

Phosphorus speciation using ion chromatography coupled with ICP-MS elucidates transformations of phosphorus compounds on reactive surfaces.

Hypophosphatasia is a rare genetic disease caused by deficient alkaline phosphatase (AP) activity. In adults, this causes functional limitations, substantial disability with pain and reduced quality of life. This Phase 1b, single-center, open-label trial investigated ilofotase alfa, a fully human recombinant protein intended as enzyme replacement therapy, in adults with hypophosphatasia. Changes in plasma levels of AP substrates inorganic pyrophosphate and pyridoxal-5'-phosphate were evaluated. Participants were randomized 1:1 to receive at 0.8 or 3.2mg/kg ilofotase alfa intravenously over 1h. Twelve participants were enrolled and completed the trial. At baseline, all participants had reduced AP activity and elevated pyridoxal-5'-phosphate. The greatest reduction in inorganic pyrophosphate and pyridoxal-5'-phosphate occurred 2h after start of dosing in both treatment groups. Across the 10-d follow-up period, inorganic pyrophosphate values returned to baseline levels more rapidly in the 0.8mg/kg group compared with the 3.2mg/kg group. Mean circulating AP activity peaked 24h after dosing and subsequently declined but remained above the lower limit of normal throughout the study. A dose-proportional increase in ilofotase alfa was observed, reaching peak concentration 1-h post-infusion. Eight treatment-emergent adverse events occurred, all classified as mild. These data demonstrate that single-dose ilofotase alfa enhances AP activity and results in dose-dependent reductions in primary disease-specific biomarkers without undesired effects on mineral homeostasis. Clinical trial registration number: ClinicalTrials.gov number: NCT05890794.

Pathological ectopic calcification of soft tissues can arise from reduced or absent levels of inorganic pyrophosphate (PPi), a key inhibitor of calcium hydroxyapatite deposition in soft connective tissues. The role of PPi in regulating mineralization has been recognized for decades, thanks to the pivotal work of Herbert Fleisch and colleagues; and its clinical relevance has been underscored by the identification of hereditary metabolic disorders, collectively termed PPi deficiency syndromes. These are caused by pathogenic variants in the essential genes for maintaining PPi homeostasis: ATP-binding cassette subfamily C member 6 (ABCC6), ectonucleotide pyrophosphate phosphodiesterase 1 (ENPP1), progressive ankylosis protein (ANK), tissue-nonspecific alkaline phosphatase (ALPL), CD73, and CD39. In recent years, abnormalities in lipid metabolism have been reported in these monogenic conditions. However, a common understanding of these alterations has yet to be established. This review provides an overview of the pathophysiology of PPi deficiency syndromes-pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to CD73 deficiency, ankylosis, and Hutchinson-Gilford progeria syndrome-highlighting the lipid metabolism alterations in cells, animal models, and patients. We explore the evidence for a potential role of PPi-regulating proteins in lipid metabolic pathways to demonstrate that lipid alterations are not coincidental but entail opportunities for future research and for potential therapeutic interventions.

ABSTRACTBackgroundTo explore the cellular behavior of stem cells derived from human exfoliated deciduous teeth (SHED) in response to inorganic pyrophosphate (PPi).Materials and MethodsSHED cells were isolated from the dental pulp tissues of human primary exfoliated teeth. Cell proliferation was examined using the MTT assay, colony‐forming unit assay, and cell cycle analysis. Cell migration was evaluated using the scratch assay. Osteogenic differentiation was assessed by the expression of osteogenic marker genes and in vitro mineral deposition. Oil Red O staining was employed to determine intracellular lipid accumulation under adipogenic differentiation. For osteoclast differentiation, TRAP staining was used. The global gene expression profile was examined by RNA sequencing analysis.ResultsPPi reduced early cell apoptosis and enhanced cell migration. PPi inhibited mineral deposition dose‐dependently and significantly reduced DSPP and BGLAP expression. The higher dose of 10 μM PPi decreased RANKL mRNA expression, while it did not influence OPG mRNA levels, resulting in the reduction of the RANKL/OPG expression ratio. Culture medium from PPi‐treated SHED reduced the number of TRAP‐positive multinucleated cells. Further, PPi inhibited CEBPA but not PPARG and LPL mRNA expression under adipogenic induction. The intracellular lipid accumulation tended to decrease in PPi‐treated conditions (10 μM). The transcriptomic profiles illustrated that PPi potentially modulated several pathways, including the metabolism of lipids, interleukin‐6, TGF‐β1, and NOTCH signaling.ConclusionPPi inhibited osteo/odontogenic, adipogenic and indirectly attenuated osteoclast differentiation by SHED. This study implicated that PPi can modulate the cellular responses of SHED.

Ossification of the Posterior Longitudinal Ligament (OPLL) and Diffuse Idiopathic Skeletal Hyperostosis (DISH) are debilitating conditions characterized by pain, stiffness, myelopathy, and impaired mobility due to progressive enthesopathies and spinal fractures. These disorders worsen with age and may lead to hemiplegia. The underlying mechanisms of these diseases remain poorly understood, and effective treatments are currently lacking. To elucidate the pathogenesis of OPLL, we conducted a prospective study involving plasma analyte measurement in 50 consecutive OPLL and 25 consecutive cervical osteoarthritic (OA) patients who presented for surgical correction within the same time frame, followed by exome sequencing of 19 genes associated with phosphate wasting and spinal ligament enthesopathy/ossification. Our study identified a significant association between OPLL and ENPP1 deficiency. Specifically, we observed that OPLL patients exhibited decreased plasma levels of inorganic pyrophosphate (PPi) while maintaining unaltered alkaline phosphatase levels. Additionally, 17% of OPLL patients harbored monoallelic pathogenic variants in ENPP1, the mammalian enzyme responsible for extracellular PPi. Using Enpp1-deficient mice (Enpp1asj) to model the condition, we discovered pathologic mineralization of the spine, long bones, and tendons, alongside increased long bone and spinal fracture risk by 17 wk of age. We further assessed the therapeutic potential of two forms of ENPP1 enzyme replacement therapies. Bone-targeted ENPP1 significantly ameliorated the spinal hyperostosis, improved or normalized spinal and long bone fragility, ameliorated tendon enthesopathies, and improved trabecular microarchitecture. Meanwhile, soluble ENPP1 prevented tendon enthesopathies, normalized cortical bone microarchitecture, and improved long bone fragility. Our findings establish a clear link between decreased plasma PPi, ENPP1 deficiency, and OPLL, unveiling additional therapeutic targets to more effectively manage this poorly treated condition.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase suppresses glycolysis and proliferation of pulmonary artery smooth muscle cells in hypoxic pulmonary hypertension via inhibition of lactate dehydrogenase A.

Effects of anthropogenic calcium enrichment and phosphorus limitation on Phaeocystis globosa bloom formation.

Tissue-nonspecific alkaline phosphatase (TNAP) is primarily known for its role in skeletal mineralization, through the hydrolysis of inorganic pyrophosphate (PPi). Here we demonstrate that TNAP-knockout mice exhibit liver steatosis and reduced serum triglyceride levels, mirroring the effects of choline deficiency, which impairs phosphatidylcholine synthesis, an essential component of VLDL. In fasting WT mice, TNAP inhibition via SBI-425 administration decreases choline levels in blood and liver. Incubating mouse or human serum with SBI-425 inhibits the dephosphorylation of phosphocholine and phosphoethanolamine, an alternative substrate for hepatic phosphatidylcholine synthesis. In hepatocytes, TNAP inhibition impedes proliferation when the medium is supplemented with phosphocholine instead of choline. Recombinant TNAP hydrolyzes phosphocholine and phosphoethanolamine with similar efficiency than PPi. X-ray diffraction and cryo-EM identified the residues in TNAP’s active site interacting with phosphocholine, PPi and the TNAP inhibitor. In summary, TNAP is the phosphatase enabling cellular choline uptake during fasting, participating in hepatic lipid metabolism.

Hypophosphatasia (HPP) is a rare, inherited metabolic bone disorder characterized by deficient activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme encoded by the ALPL gene. Loss-of-function mutations in ALPL lead to reduced TNAP activity, resulting in the accumulation of extracellular inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation and skeletal mineralization. Clinically, this presents as a broad spectrum of dento-osseous complications, including premature tooth loss, rickets or osteomalacia, and calcific arthropathies.

Disclosure: B. Singh: None. A.P. Amblee: None.Hypophosphatasia (HPP) is a rare bone mineralization disease which can present from childhood to adulthood. In the United States, it affects approximately 500-600 individuals per year. HPP is caused by inactivating mutations in the ALPL gene leading to buildup of inorganic pyrophosphate and impaired bone mineralization. Although generalized body pain, skeletal deformities, and dental abnormalities are the most common presenting features, approximately 20% of adults with HPP can present with renal abnormalities. Within this subgroup, 14% can present with hypercalcemia, hyperphosphatemia, or hypercalciuria. This case report is unique as the only feature found in a patient diagnosed with HPP was isolated hypercalciuria. A 51 year old male with HIV and chronic vitamin D deficiency was referred management of vitamin D deficiency . He was on bictegravir, emtricitabine, and tenofovir alafenamide for 14 years. He had no history of dental problems. He had a previous fracture of the tibia and wrist from a traumatic fall in his late 20s. Labs showed vitamin D was 19.78 ng/ml (normal range 30-100 ng/ml) , corrected calcium 8.9 mg/dl (normal range 8.7-10.4 mg/dl), alkaline phosphatase (AP) 40 U/L (normal range 45-117 U/L), and PTH 72 pg/mL (normal range 18-80.1 pg/ml). Thyroid studies and testosterone levels were within normal range. DXA scan showed normal bone mineral and low FRAX score. Vitamin D3 dosing was increased from 2000 to 5000 units daily. A secondary workup showed normal serum and urine protein electrophoresis, intact PTH, and celiac panel. He had an elevated 24-hour urine calcium level of 451 mg/24 hr. Due to the hypercalciuria, he was started on low dose hydrochlorothiazide. Vitamin D level normalized in 3 months ( 33 ng/ml)and AP remained low (38 U/L). Genetic testing for hypophosphatasia showed he was heterozygous for pathogenic variant in ALPL resulting in amino acid substitution p.Ala176Thr. Since this patient did not have any history of fractures, calcium and vitamin D supplementation was recommended. The unique feature is that he only presented with hypercalciuria which can have a long term risk for osteoporosis, kidney stone and chronic kidney disease . Due to heterogeneity in the presentation and disease course, HPP can be missed or underdiagnosed. Nonskeletal findings may be the main presenting factor in some adults, such as hypercalciuria in this case. Although more studies are required to look at the prevalence of isolated hypercalciuria in adult HPP patients, renal calcium abnormalities can be found in one fifth of HPP cases. HPP has a significantly negative effect on quality of life and early diagnosis with multidisciplinary interventions can help improve patient outcomes long term.Presentation: Sunday, July 13, 2025

X-linked hypophosphatemia (XLH) is caused by mutations in the PHEX gene, which leads to increased levels of fibroblast growth factor 23 and hypophosphatemia, contributing to rickets, osteomalacia, and dentoalveolar defects, including severe dentin hypomineralization, thin cementum, and alveolar bone osteomalacia. Current XLH treatment options appear to have limited efficacy on dentoalveolar tissues, suggesting underlying disease mechanisms that remain unchecked. Increased production of inorganic pyrophosphate (PPi) and osteopontin (OPN), both mineralization inhibitors, has been posited to contribute to mineralization defects in XLH. The enzyme, tissue-nonspecific alkaline phosphatase (TNAP) reduces PPi levels via hydrolysis and inactivates OPN by dephosphorylation. Our previous study showed improved alveolar bone socket healing in Hyp mice administered mineralized tissue-targeted TNAP (TNAP-Fc-D10). We hypothesized that increased TNAP would partially ameliorate developmental mineralization defects in XLH by dually reducing PPi levels and dephosphorylating and inactivating OPN. In a proof-of-principle study to investigate pathological mechanisms, we delivered systemic (subcutaneous injection) and local (submucosal injection to mandibles) TNAP-Fc-D10 injections to the Hyp mutant mouse model of XLH from 7 to 60 d postnatal (dpn). While systemic delivery was ineffective at improving dentin or bone properties, micro-CT and histology analyses demonstrated that local delivery of TNAP-Fc-D10 increased dentin thickness, root length, alveolar bone volume, alveolar bone proper (ABP) volume and density, PDL attachment, and acellular cementum thickness, compared to control Hyp mice receiving a sham injection. Dynamic mechanical testing confirmed partially improved mechanical properties in locally treated vs untreated Hyp mice, suggesting incompletely improved periodontal function. Quantitative PCR revealed increased Dspp expression in molars of treated Hyp mice. In conclusion, we found TNAP administration reduced dentoalveolar defects in Hyp mice when delivered locally into dentoalveolar structures, proof-of-principle pointing to a pathological contribution by PPi and/or OPN and highlighting a promising adjunctive approach considering limitations of current treatment modalities.