ALPL regulates pro-angiogenic capacity of mesenchymal stem cells through ATP-P2X7 axis controlled exosomes secretion.

BACKGROUND: Early-onset bone dysplasia is a common manifestation of hypophosphatasia (HPP), an autosomal inherited disease caused by ALPL mutation. ALPL ablation induces prototypical premature bone ageing characteristics, resulting in impaired osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMMSCs). As angiogenesis is tightly coupled with osteogenesis, it also plays a necessary role in sustaining bone homeostasis. We have previously observed a decrease in expression of angiogenesis marker gene CD31 in the metaphysis of long bone in Alpl+/- mice. However, the role of ALPL in regulation of angiogenesis in bone has remained largely unknown. METHODS: Exosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively. RESULTS: We verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs. CONCLUSION: The ALPL-ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL-deficient bone defects.

Use of Complementary and Alternative Medicine in Patients with Rare Bone Diseases and Osteoporosis.

(1) Background: The use of complementary and alternative medicine (CAM) has seen a notable increase in popularity. However, there is an absence of data regarding the prevalence of CAM use in patients with rare bone diseases (RBDs). (2) Methods: This monocentric, cross-sectional study was carried out in a reference hospital for RBDs. RBD patients included individuals with osteogenesis imperfecta, hypophosphatasia and X-linked hypophosphatemia, and their data were compared with those of patients with osteoporosis (OPO) and of healthy controls (CON). This study utilized the German version (I-CAM-G) of the I-CAM questionnaire. (3) Results: This study comprised 50 RBD patients [mean age (SD) of 48.8 (±15.9), 26% male], 51 OPO patients [66.6 (±10.0), 9.8% male] and 52 controls [50.8 (±16.3), 26.9% male]. Treatments by naturopaths/healers were more prevalent in the RBD group (11.4%) compared with OPO (0%) and CON (5.8%) (p = 0.06). More than half of the OPO (60.8%) and CON (63.5%) patients and 46% of the RBD patients reported vitamin/mineral intake within the past 12 months (p = 0.16). Individuals with tertiary education had a significantly higher odds ratio of 2.64 (95% CI: 1.04-6.70, p = 0.04) for visiting any CAM provider. Further, OPO patients were significantly less likely to use self-help techniques compared with the CON group (OR = 0.42, 95% CI: 0.19-0.95; p = 0.04). (4) Conclusions: Herbal medicine, vitamin and mineral supplements, and self-help techniques were the most common forms of CAM reported by patients with RBDs. However, the use of CAM was generally low.

A Case of Hypophosphatasia With Normal Alkaline Phosphatase Levels.

Background/Objective: Hypophosphatasia (HPP) is a rare disease associated with low serum alkaline phosphatase (ALP) activity. Here, we present a case of a patient with normal serum ALP levels diagnosed with HPP. Case Report: A 36-year-old woman presented with progressive fatigue, weakness, and joint pain. She had been evaluated in the past for genetic disorders due to these symptoms and was found to have a history of several total ALP levels within normal limits but elevated vitamin B6 levels. She also reported having loose teeth and "gray gums" during her childhood. Bone-specific ALP was tested for suspicion of HPP and returned at 4.4 µ/L (reference range, 5.3-19.5 µg/L), which prompted genetic testing. Genetic testing confirmed a positive pathogenetic variant of the ALPL gene, the c.542C>T (p.Ser181Leu) variant. She started asfotase alfa treatment to improve her symptoms. Discussion: HPP was diagnosed based on clinical suspicion supported by laboratory findings, which can cause it to be underdiagnosed or misdiagnosed. Current literature reports that a low total ALP level is the main biochemical marker of HPP and the only level needed to diagnose the disease. However, bone-specific ALP, a common marker used for bone turnover, has not been required to be tested. Conclusion: This case highlights a patient with normal total ALP, but low bone-specific ALP diagnosed with HPP confirmed by genetic testing. This case warrants future investigation into the diagnostic approach to HPP and the diagnostic utility between ALP and bone-specific ALP.

A case report of odonto-hypophosphatasia with a novel variant in the ALPL gene.

OBJECTIVES: Hypophosphatasia (HPP) is a rare skeletal dysplasia caused by variants in the alkaline phosphatase (ALPL) gene. More than 400 pathogenic variants of the ALPL gene have been registered in the ALPL gene variant database. Here, we describe the case of a Japanese child with odonto-hypophsphatasia (odonto-HPP) and a novel ALPL variant. CASE PRESENTATION: At the age of 2 years and 1 month, he prematurely lost one deciduous tooth, with the root intact, when he fell and hit his face lightly. Three months later, he lost another adjacent deciduous tooth without incentive. His serum alkaline phosphatase (ALP) level was 72 U/L. His urine phosphoethanolamine (PEA) level was extremely high at 938 µmol/mg·Cre. The serum pyridoxal 5'-phosphaye (PLP) level was 255.9 nmol/L. Based on the clinical symptoms and laboratory findings, the patient was clinically diagnosed with odonto-HPP. Genetic analysis of the ALPL gene revealed a heterozygous variant (NM_000478.6:c.1151C>A, p.Thr384Lys). CONCLUSIONS: We report a case of odonto-HPP with a novel variant in the ALPL gene. HPP is a rare disease, and the heterozygous mutation in the ALPL gene highlights the novelty of this case.

Pyridoxine challenge reflects pediatric hypophosphatasia severity and thereby examines tissue-nonspecific alkaline phosphatase's role in vitamin B6 metabolism.

Alkaline phosphatase (ALP) is detected in most human tissues. However, ALP activity is routinely assayed using high concentrations of artificial colorimetric substrates in phosphate-free laboratory buffers at lethal pH. Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the ALP isoenzyme expressed in bone, liver, kidney, and elsewhere and is therefore designated "tissue-nonspecific" ALP (TNSALP). Consequently, HPP harbors clues concerning the biological function of this phosphohydrolase that is anchored onto the surface of cells. The biochemical signature of HPP features low serum ALP activity (hypophosphatasemia) together with elevated plasma levels of three natural substrates of TNSALP: i) phosphoethanolamine (PEA), a component of the linkage apparatus that binds ALPs and other proteins to the plasma membrane surface; ii) inorganic pyrophosphate (PPi), an inhibitor of bone and tooth mineralization; and iii) pyridoxal 5'-phosphate (PLP), the principal circulating vitameric form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving several hundred ALPL mutations underlies the remarkably broad-ranging expressivity of HPP featuring tooth loss often with muscle weakness and rickets or osteomalacia. Thus, HPP associates the "bone" isoform of TNSALP with biomineralization, whereas the physiological role of the "liver", "kidney", and other isoforms of TNSALP remains uncertain. Herein, to examine HPP's broad-ranging severity and the function of TNSALP, we administered an oral challenge of pyridoxine (PN) hydrochloride to 116 children with HPP. We assayed both pre- and post-challenge serum ALP activity and plasma levels of PLP, the B6 degradation product pyridoxic acid (PA), and the B6 vitamer pyridoxal (PL) that can enter cells. Responses were validated by PN challenge of 14 healthy adults and 19 children with metabolic bone diseases other than HPP. HPP severity was assessed using our HPP clinical nosology and patient height Z-scores. PN challenge of all study groups did not alter serum ALP activity in our clinical laboratory. In HPP, both the post-challenge PLP level and the PLP increment correlated (Ps < 0.0001) with the clinical nosology and height Z-scores (Rs = +0.6009 and + 0.4886, and Rs = -0.4846 and - 0.5002, respectively). In contrast, the plasma levels and increments of PA and PL from the PN challenge became less pronounced with HPP severity. We discuss how our findings suggest extraskeletal TNSALP primarily conditioned the PN challenge responses, and explain why they caution against overzealous B6 supplementation of HPP.

Impressive clinical improvement and disappearance of neuropathic pain in an adult patient with hypophosphatasia treated with asfotase alfa.

Hypophosphatasia (HPP) is a rare disorder, resulting from loss-of-function variants of the ALPL gene encoding non-tissue specific alkaline phosphatase (TNSALP). Presentation varies largely, with increased severity usually occurring with earlier disease onset. Here we describe the clinical improvement of a 57-year-old woman with childhood onset HPP, after initiating treatment with asfotase alfa (Strensiq®). This was started because of the rapid and progressive radiological deterioration of bone structure after placement of nails in both upper legs for spontaneous atypical femur fracture (AFF) - like fractures. Initiation of treatment, not only resulted in stabilization of bone structure on X-rays, but within a few weeks there was a dramatic reduction of burning pain sensations in the lower legs, attributed in retrospect to neuropathic pain, and also almost complete disappearance of headaches. Additionally, unhealed metatarsal fractures finally healed after almost 10 years. Drug efficacy was further evaluated through -quality of life questionnaires and multiple tests conducted by the physiotherapist, and showed clear improvements. Within 3 months after starting asfotase alfa, the patient was able to carry out her daily tasks indoors without relying on a walker and even started electric bike rides for 20 km/day. In conclusion, treatment with asfotase alfa, halted rapid radiological bone deterioration after bilateral intramedullary femoral pen placement and strongly increased quality of life, marked by rapid disappearance of neuropathic pain, reduction in headaches and musculoskeletal pains, and enhanced muscle strength and mobility. The quick and almost complete disappearance of neuropathic pain and headache suggests a relation with disturbed levels of metabolites in HPP.

Case Report of a 4-Year-Old Girl with Stage IV Grade C Localized Periodontitis (Pre-Puberal Localized Aggressive Periodontitis) Affected by Misrecognition and Late Diagnosis.

BACKGROUND AND OBJECTIVES: Stage IV grade C localized periodontitis (pre-puberal localized aggressive periodontitis/LPP) is a rare form of inflammatory periodontal disease occurring in clinically healthy individuals (no/small calculus/dental plaque traces), due a hyper-aggressive auto-immune response to high amounts of bacteria present in the oral cavity. CASE PRESENTATION: This case report describes a 4-year-old Caucasian girl with localized gingival inflammation and advanced bone loss around the temporary lower left canine. The first diagnostic assumption was hypophosphatasia, and the patient was sent for further genetic and metabolic investigations (which turned out to be negative). The LPP diagnosis was made during the family's summer holidays due to her parents' concerns about persistent gingival inflammation and tooth mobility. RESULTS: The diagnosis of LPP was supported by clinical oral examination results, earlier X-rays, earlier blood tests, and a periodontal bacterial test. The treatment was limited to avoid spreading inflammation to other teeth (via topical antibiotic treatment) due to our limited time frame, while the main problem of excessive amounts of periodontal bacteria in the oral cavity was not addressed. The tooth was eventually lost. CONCLUSIONS: The ability to early recognize radiological and clinical LPP signs correlated with understanding of its pathological auto-immune mechanism is extremely important for expanding treatment options, since bone preservation and reducing amounts of bacteria are strictly correlated with therapeutic speed.

A Delphi panel to build consensus on assessing disease severity and disease progression in adult patients with hypophosphatasia in the United States.

BACKGROUND: Hypophosphatasia (HPP) is an inborn error of metabolism with a variable presentation. We conducted a modified Delphi panel to obtain expert consensus on knowledge gaps regarding disease severity and progression in adult patients with HPP. METHODS: Healthcare professionals (HCPs) with experience managing adult patients with HPP were recruited to participate in a 3-round Delphi panel (round 1: paper survey and 1:1 interview; rounds 2-3: email survey). Panelists rated the extent of their agreement with statements about disease severity and progression in adult patients with HPP. Consensus was defined as ≥ 80% agreement. RESULTS: Ten HCPs completed round 1; nine completed rounds 2 and 3. Consensus was reached on 46/120 statements derived from steering committee input. Disease severity markers in adult patients with HPP can be bone-related (recurrent/poorly healing fractures, pseudo-fractures, metatarsal fractures, osteomalacia) or involve dentition or physiologic/functional manifestations (use of mobility devices/home modifications, abnormal gait, pain). Disease progression markers can include recurrent/poorly healing low-trauma fractures, development of ectopic calcifications, and/or impairment of functional activity. Panelists supported the development of a tool to help assess disease severity in the clinic and track changes in severity over time. Panelists also highlighted the role of a multidisciplinary team, centers with expertise, and the need to refer patients when disease severity is not clear. CONCLUSIONS: These statements regarding disease severity, progression, and assessment methods address some knowledge gaps in adult patients with HPP and may be helpful for treating HCPs, although the small sample size affects the ability to generalize the healthcare provider experience.

Proposed diagnostic criteria for the diagnosis of hypophosphatasia in children and adolescents: results from the HPP International Working Group.

Hypophosphatasia (HPP) is a rare inborn error of metabolism that presents variably in both age of onset and severity. HPP is caused by pathogenic variants in the ALPL gene, resulting in low activity of tissue nonspecific alkaline phosphatase (TNSALP). Patients with HPP tend have a similar pattern of elevation of natural substrates that can be used to aid in diagnosis. No formal diagnostic guidelines currently exist for the diagnosis of this condition in children, adolescents, or adults. The International HPP Working Group is a comprised of a multidisciplinary team of experts from Europe and North America who have expertise in the diagnosis and management of patients with HPP. This group reviewed 93 papers through a Medline, Medline In-Process, and Embase search for the terms "HPP" and "hypophosphatasia" between 2005 and 2020 and that explicitly address either the diagnosis of HPP in children, clinical manifestations of HPP in children, or both. Two reviewers independently evaluated each full-text publication for eligibility and studies were included if they were narrative reviews or case series/reports that concerned diagnosis of pediatric HPP or included clinical aspects of patients diagnosed with HPP. This review focused on 15 initial clinical manifestations that were selected by a group of clinical experts.The highest agreement in included literature was for pathogenic or likely pathogenic ALPL variant, elevation of natural substrates, and early loss of primary teeth. The highest prevalence was similar, including these same three parameters and including decreased bone mineral density. Additional parameters had less agreement and were less prevalent. These were organized into three major and six minor criteria, with diagnosis of HPP being made when two major or one major and two minor criteria are present.

The challenge of hypophosphatasia diagnosis in adults: results from the HPP International Working Group Literature Surveillance.

Hypophosphatasia (HPP) is an inborn error of metabolism caused by reduced or absent activity of the tissue non-specific alkaline phosphatase (TNSALP) enzyme, resulting from pathogenic variants in the ALPL gene. Clinical presentation of HPP is highly variable, including lethal and severe forms in neonates and infants, a benign perinatal form, mild forms manifesting in adulthood, and odonto-HPP. Diagnosis of HPP remains a challenge in adults, as signs and symptoms may be mild and non-specific. Disease presentation varies widely; there are no universal signs or symptoms, and the disease often remains underdiagnosed or misdiagnosed, particularly by clinicians who are not familiar with this rare disorder. The absence of diagnosis or a delayed diagnosis may prevent optimal management for patients with this condition. Formal guidelines for the diagnosis of adults with HPP do not exist, complicating efforts for consistent diagnosis. To address this issue, the HPP International Working Group selected 119 papers that explicitly address the diagnosis of HPP in adults through a Medline, Medline In-Process, and Embase search for the terms "hypophosphatasia" and "HPP," and evaluated the pooled prevalence of 17 diagnostic characteristics, initially selected by a group of HPP clinical experts, in eligible studies and in patients included in these studies. Six diagnostic findings showed a pooled prevalence value over 50% and were considered for inclusion as major diagnostic criteria. Based on these results and according to discussion and consideration among members of the Working Group, we finally defined four major diagnostic criteria and five minor diagnostic criteria for HPP in adults. Authors suggested the integrated use of the identified major and minor diagnostic criteria, which either includes two major criteria, or one major criterion and two minor criteria, for the diagnosis of HPP in adults.

Hypophosphatasia diagnosis: current state of the art and proposed diagnostic criteria for children and adults.

BACKGROUND: This manuscript provides a summary of the current evidence to support the criteria for diagnosing a child or adult with hypophosphatasia (HPP). The diagnosis of HPP is made on the basis of integrating clinical features, laboratory profile, radiographic features of the condition, and DNA analysis identifying the presence of a pathogenic variant of the tissue nonspecific alkaline phosphatase gene (ALPL). Often, the diagnosis of HPP is significantly delayed in both adults and children, and updated diagnostic criteria are required to keep pace with our evolving understanding regarding the relationship between ALPL genotype and associated HPP clinical features. METHODS: An International Working Group (IWG) on HPP was formed, comprised of a multidisciplinary team of experts from Europe and North America with expertise in the diagnosis and management of patients with HPP. Methodologists (Romina Brignardello-Petersen and Gordon Guyatt) and their team supported the IWG and conducted systematic reviews following the GRADE methodology, and this provided the basis for the recommendations. RESULTS: The IWG completed systematic reviews of the literature, including case reports and expert opinion papers describing the phenotype of patients with HPP. The published data are largely retrospective and include a relatively small number of patients with this rare condition. It is anticipated that further knowledge will lead to improvement in the quality of genotype-phenotype reporting in this condition. CONCLUSION: Following consensus meetings, agreement was reached regarding the major and minor criteria that can assist in establishing a clinical diagnosis of HPP in adults and children.

The Global ALPL gene variant classification project: Dedicated to deciphering variants.

BACKGROUND: Hypophosphatasia (HPP) is an inherited multisystem disorder predominantly affecting the mineralization of bones and teeth. HPP is caused by pathogenic variants in ALPL, which encodes tissue non-specific alkaline phosphatase (TNSALP). Variants of uncertain significance (VUS) cause diagnostic delay and uncertainty amongst patients and health care providers. RESULTS: The ALPL gene variant database (https://alplmutationdatabase.jku.at/) is an open-access archive for interpretation of the clinical significance of variants reported in ALPL. The database contains coding and non-coding variants, including single nucleotide variants, insertions/deletions and structural variants affecting coding or non-coding sequences of ALPL. Each variant in the database is displayed with details explaining the corresponding pathogenicity, and all reported genotypes and phenotypes, including references. In 2021, the ALPL gene variant classification project was established to reclassify VUS and continuously assess and update genetic, phenotypic, and functional variant information in the database. For this purpose, the database provides a unique submission system for clinicians, geneticists, genetic counselors, and researchers to submit VUS within ALPL for classification. An international, multidisciplinary consortium of HPP experts has been established to reclassify the submitted VUS using a multi-step process adhering to the stringent ACMG/AMP variant classification guidelines. These steps include a clinical phenotype assessment, deep literature research including artificial intelligence technology, molecular genetic assessment, and in-vitro functional testing of variants in a co-transfection model to measure ALP residual activity. CONCLUSION: This classification project and the ALPL gene variant database will serve the global medical community, widen the genotypic and phenotypic HPP spectrum by reporting and characterizing new ALPL variants based on ACMG/AMP criteria and thus facilitate improved genetic counseling and medical decision-making for affected patients and families. The project may also serve as a gold standard framework for multidisciplinary collaboration for variant interpretation in other rare diseases.

Mild hypophosphatasia may be twice as prevalent as previously estimated: an effective clinical algorithm to detect undiagnosed cases.

OBJECTIVES: Since the prevalence of hypophosphatasia (HPP), a rare genetic disease, seems to be underestimated in clinical practice, in this study, a new diagnostic algorithm to identify missed cases of HPP was developed and implemented. METHODS: Analytical determinations recorded in the Clinical Analysis Unit of the Hospital Universitario Clínico San Cecilio in the period June 2018 - December 2020 were reviewed. A new clinical algorithm to detect HPP-misdiagnosed cases was used including the following steps: confirmation of persistent hypophosphatasemia, exclusion of secondary causes of hypophosphatasemia, determination of serum pyridoxal-5'-phosphate (PLP) and genetic study of ALPL gene. RESULTS: Twenty-four subjects were selected to participate in the study and genetic testing was carried out in 20 of them following clinical algorithm criteria. Eighty percent of patients was misdiagnosed with HPP following the current standard clinical practice. Extrapolating these results to the current Spanish population means that there could be up to 27,177 cases of undiagnosed HPP in Spain. In addition, we found a substantial proportion of HPP patients affected by other comorbidities, such as autoimmune diseases (∼40 %). CONCLUSIONS: This new algorithm was effective in detecting previously undiagnosed cases of HPP, which appears to be twice as prevalent as previously estimated for the European population. In the near future, our algorithm could be globally applied routinely in clinical practice to minimize the underdiagnosis of HPP. Additionally, some relevant findings, such as the high prevalence of autoimmune diseases in HPP-affected patients, should be investigated to better characterize this disorder.

A Novel Case of Concomitant PHEX and ALPL Mutation In a Family With Rickets.

Currently, no published cases report concomitant X-linked hypophosphatemia (XLH) and adult hypophosphatasia (HPP). Both diseases share clinical phenotypes that are almost indistinguishable. The correct diagnosis may be missed without a standardized laboratory and genetic testing approach. Pathogenic variants in the phosphate regulating endopeptidases homolog X-linked gene (PHEX) and the tissue-nonspecific alkaline phosphatase gene (ALPL) are genes that cause XLH and HPP, respectively. We describe a concomitant yet undescribed genetic pathogenic variant in a family. A 61-year-old woman was referred by orthopedic surgery for the presence of bilateral leg bowing and short stature during the assessment of knee surgery. The patient had a biochemical workup relevant for low serum phosphorus and 1,25-dihydroxy vitamin D and normal alkaline phosphatase (ALP). Genetic analysis revealed pathogenic variants in PHEX and ALPL. Her 42-year-old daughter shared identical symptoms and genetic variants with her mother. Both patients started conventional treatment for XLH with phosphorus and vitamin D, and the daughter later switched to burosumab-twza. Adult XLH and HPP may have similarities in clinical presentation but differ in some essential laboratory findings. Normal ALP levels helped direct our diagnosis toward XLH. However, the diagnosis was challenging due to the presence of concurrent variants in the genes involved. These variants illustrate the significant heterogeneity of the clinical expression.

Impact of Restricted Phosphorus, Calcium-adjusted Diet on Musculoskeletal and Mental Health in Hypophosphatasia.

Context: Impairments in musculoskeletal and mental health are common in adults with Hypophosphatasia (HPP). Restricted phosphorus intake has been suggested to positively affect symptoms in HPP, but there is a lack of interventional evidence. Objective: This work aimed to evaluate the effect of a phosphorus-restricted, calcium-adjusted diet on musculoskeletal and mental health in HPP. Methods: A prospective, noncontrolled, single-center interventional study (NuSTEPS II) was conducted among outpatients at the Osteology Department, University of Wuerzburg, Germany. A total of 26 adults with an established HPP diagnosis received a standardized diet with a defined daily intake of phosphorus (1160-1240 mg/d) and calcium (870-930 mg/d) over 8 weeks. Main outcome measures were functional testing and patient-reported outcome measures. Results: At 8 weeks, significant improvements were observed in usual gait speed (P = .028) and the chair-rise test (P = .019), while no significant changes were seen in the 6-minute walk test (P = .468) and the timed up-and-go test (P = .230). Pain was not significantly reduced according to the visual analog scale (VAS) (P = .061), pain subscale of the 36-Item Short-Form Health Survey (SF-36) (P = .346), and Pain Disability Index (P = .686). Further, there was a significant improvement in the SF-36 vitality subscale (P = .022) while all other subscales as well as the Lower Extremity Functional Scale (P = .670) and the Fatigue Assessment Scale (P = .392) did not change significantly. Adjustments of mineral intake were not associated with relevant alterations regarding the intake of energy and energy-supplying nutrients or body composition. Conclusion: Adjusting phosphorus and calcium intake may positively affect individual symptoms in adults with HPP, but overall clinical effectiveness regarding major issues like pain and endurance appears limited.

Compromised Muscle Properties in a Severe Hypophosphatasia Murine Model.

Hypophosphatasia (HPP) is a rare metabolic bone disorder characterized by low levels of tissue non-specific alkaline phosphatase (TNAP) that causes under-mineralization of the bone, leading to bone deformity and fractures. In addition, patients often present with chronic muscle pain, reduced muscle strength, and an altered gait. In this work, we explored dynamic muscle function in a homozygous TNAP knockout mouse model of severe juvenile onset HPP. We found a reduction in skeletal muscle size and impairment in a range of isolated muscle contractile properties. Using histological methods, we found that the structure of HPP muscles was similar to healthy muscles in fiber size, actin and myosin structures, as well as the α-tubulin and mitochondria networks. However, HPP mice had significantly fewer embryonic and type I fibers than wild type mice, and fewer metabolically active NADH+ muscle fibers. We then used oxygen respirometry to evaluate mitochondrial function and found that complex I and complex II leak respiration were reduced in HPP mice, but that there was no disruption in efficiency of electron transport in complex I or complex II. In summary, the severe HPP mouse model recapitulates the muscle strength impairment phenotypes observed in human patients. Further exploration of the role of alkaline phosphatase in skeletal muscle could provide insight into mechanisms of muscle weakness in HPP.

Hypophosphatasia: presentation and response to asfotase alfa.

Hypophosphatasia (HPP) is a rare bone disease with limited scientific evidence on the tolerability and safety of its novel treatment, Asfotase Alfa (AA). We report 7 HPP patients' heterogenous presentations and the significant improvement in various clinical outcomes attained with AA shedding light on this highly effective and safe therapy. INTRODUCTION: Hypophosphatasia (HPP) is a rare inherited metabolic bone disorder characterized by a deficiency in the tissue non-specific alkaline phosphatase (TNSALP) due to loss of function mutation in the ALPL gene. HPP is associated with impaired skeletal mineralization due to elevations in inorganic pyrophosphate and altered phosphate : pyrophosphate ratio. Asfotase alfa (AA) "enzyme replacement" was approved for treatment of HPP in 2015. We present 7 patients with HPP, 5 with pediatric-onset, and 2 with adult-onset, who have been treated with AA and describe the efficacy and safety in these patients. METHODS: 7 patients (4 females, 3 males) aged 19-68 years with HPP were included in this study. Diagnosis of HPP was confirmed by DNA analysis. AA was administered in doses of 6mg/kg/week with a mean follow-up of 6 months (SD= 5). RESULTS: Subjective improvement in muscle strength, muscle pain, walking ability, and walking distance with a reduction in the use of gait aids was seen "with AA in HPP patients." Muscle strength and pain improved by up to 70% from baseline as quantified subjectively by patients. Walking distance improved by up to 100%. Patients also reported improved cognition, mood, and energy levels, with up to 90% improvement in mood and 75% improvement in energy levels. 4 out of 6 patients first noted clinical signs of improvement after 3 months of being on therapy. 1 out of the 7 patients sustained a toe fracture 10 months from being on AA. AA was well-tolerated with injection site reactions being the most reported adverse effect. CONCLUSION: HPP treatment with AA in individuals with both pediatric and adult-onset forms resulted in significant subjective improvement in musculoskeletal and cognitive manifestations in addition to patients' quality of life. The drug was well tolerated in 6 patients. 1 patient discontinued therapy because of minor adverse effects with myalgias.

Cranial Neural Crest Specific Deletion of Alpl (TNAP) via P0-Cre Causes Abnormal Chondrocyte Maturation and Deficient Cranial Base Growth.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.

First Reported Case of a Pyrophosphate Kidney Stone in a Human.

Urolithiasis composed of pyrophosphate salts has only been reported in animals, in the form of potassium magnesium pyrophosphate. However, there have been no reports of pyrophosphate stones in humans. Hypophosphatasia is an inherited disease characterized by low alkaline phosphatase activity and elevated levels of pyrophosphate in blood and urine. Urolithiasis is a part of the hypophosphatasia phenotype. The role of elevated urine pyrophosphate levels in the formation of stones in hypophosphatasia is unknown. Here, we report a case of a 60-year-old man with recurrent urolithiasis. The patient's most recent presentation was gross hematuria and his computed tomography scan showed bilateral kidney stones. Stones were removed via retrograde intrarenal surgery. Stone analysis revealed a composition of potassium magnesium pyrophosphate. The patient also has a long history of fracturing bone disease which led to the consideration of hypophosphatasia as the cause of both his bone disease and pyrophosphate stones. Hypophosphatasia was confirmed by genetic analysis. Pyrophosphate has been of interest in the fields of mineral metabolism because of its action as a crystallization inhibitor. However, pyrophosphate at elevated concentrations in the presence of divalent cations can exceed its solubility. Nephrocalcinosis and stone disease have been described in hypophosphatasia; stones have been assumed to be calcium phosphate but no compositional analysis has been reported. This is the first report of human stones composed of pyrophosphate salts, which led to the subsequent diagnosis of hypophosphatasia in this patient.

Serum alkaline phosphatase can be elevated in patients with hypophosphatasia due to liver disease.

BACKGROUND: Hypophosphatasia (HPP) is a rare inherited disorder caused by pathogenic loss-of-function variants in the ALPL gene, encoding the tissue-nonspecific isoenzym of alkaline phosphatase (ALP; TNSALP). Low serum ALP is the biochemical hallmark of HPP, but it is unknown whether ALP levels can increase due to concurring liver disease, which may lead to a missed diagnose of HPP. We present a patient with genetically confirmed HPP, who showed a transient increase of serum ALP levels due to alcohol-induced hepatitis. CLINICAL REPORT: A 71-year old man was seen at our Bone Center for surveillance of HPP. Serum ALP was always low (23 U/L; reference value: <115 U/L). During follow-up, his serum ALP increased (156 U/L, further rising to 204 U/L), with concomitantly elevated serum gamma-glutamyl transferase and transaminases, and a rise in bone specific ALP (18.7 µg/L; reference value: 5.7-32.9 µg/L). This was attributed to alcohol-induced hepatitis. After refraining from alcohol intake, both serum ALP and bone specific ALP levels returned to initial low levels (30 U/L and 4.3 µg/L respectively). CONCLUSIONS: We demonstrated the history of a 71-year old patient with HPP, presenting during routine follow-up with an elevated serum ALP level up to 204 U/L due to alcohol-induced hepatitis. This case illustrates that the diagnosis of HPP can potentially be missed when ALP levels are normal or elevated due to a concomitant liver disease.