ALPL Research Articles

Musculoskeletal pain and muscular weakness as the main symptoms of adult hypophosphatasia in a Spanish cohort: clinical characterization and identification of a new ALPL gene variant.

Hypophosphatasia (HPP) is a rare inherited disorder, caused by mutations in the alkaline phosphatase (ALPL) gene, which encodes for the tissue non-specific alkaline phosphatase (TNSALP) isoform of alkaline phosphatase (ALP). Adult HPP is one of the mild forms that presents with unspecific signs such as osteopenia, osteomalacia and muscle involvement. Our purpose was to identify and characterize possibly misdiagnosed adult HPP patients at a clinical and biochemical level. At the laboratory of Miguel Servet University Hospital we retrospectively reviewed serum ALP levels in adults over a 48-month period. The clinical records of individuals with consistently low ALP levels were reviewed to exclude secondary causes. Those with persistent hypophosphatasemia were screened for symptoms of HPP. The study participants were evaluated at biochemical and genetic levels. We identified 705 ALP determinations (out of 384,000 processed) in 589 patients below the reference range (30 U/l). Only 21 patients with clinical signs and symptoms of HPP were selected for genetic testing. Finally, only 12 patients participated in the study, 83.3% of whom (10/12) harbored a pathogenic or likely pathogenic variant in a heterozygous state. The major symptoms of our cohort were the presence of musculoskeletal pain (100% of patients) and muscular weakness (83.3% patients). Mild HPP patients presenting with diffuse symptoms such as musculoskeletal pain may be undiagnosed or misdiagnosed as osteoporosis patients by routine diagnosis. It is important to identify these individuals, to avoid inappropriate treatment with antiresorptive drugs.

Treatment of hypophosphatasia

ypophosphatasia is a systemic metabolic disorder due to genetically determined deficient activity of the tissue non-specific alkaline phosphatase (TNAP). The phenotypic presentation is characterized by a wide spectrum of clinical manifestations regarding both, affected body systems and organs as well as the severity of associated deficits. Appropriate treatment strategies thus have to be multimodal in order to cover individual disease manifestation. For patients with disease onset before adulthood, enzyme replacement therapy with asfotase alfa is approved in Europe to treat the bone manifestations of the disease. Available data from clinical trials as well as real-word evidence confirm encouraging results of this treatment in severely affected children with substantial improvement regarding radiographic and functional outcome parameters as well as overall survival. In adult patients with disease manifestation pursuant to the label, published results also report substantial amelioration of disease-specific deficits along with functional improvements. Meanwhile, there is are also data supporting the safety and efficacy of long-term treatment with asfotase alfa over several years. While inflammatory muskuloskeletal pain - seemingly the most prevalent clinical manifestation along with exhaustion - can transiently be mitigated with on-demand NSARs, essential treatment options to causatively overcome that issue are still lacking. Accordingly, maintenance of musculoskeletal health and functionality requires sustained supportive treatment including physiotherapy and individually adjusted technical orthopedic support. The use and potential clinical impact of phosphate and vitamin B6 on the course of the disease requires further investigation. Current data regarding the use of bone-targeted compounds established for osteoporosis is critical in terms of antiresorptive, while osteoanabolic treatment strategies appear feasible. Considering further organ manifestation including orodental, gastrointestinal and neurological symptoms etc., the entirety of therapeutic measures should be coordinated among a multidisciplinary team and overlooked at an experienced center, while individual tasks can preferably be accomplished at local facilities near the patient’s home.

Hypophosphatasia: from birth to adulthood.

Hypophosphatasia (HPP) is an inherited disease caused by a low activity of tissue-nonspecific alkaline phosphatase, a hydrolase that removes phosphate groups from many molecules. Decreased alkaline phosphatase activity leads to the accumulation of three main metabolites, i.e., pyridoxal 5´-phosphate (PLP), inorganic pyrophosphate (PPi), and phosphoethanolamine. Impairment in PLP dephosphorylation induces seizures, while PPi accumulation inhibits bone mineralization. Clinically, HPP has a wide spectrum of presentations, ranging from neonatal death to an apparent lack of symptoms. This disease is classified into six subtypes according to the age at onset of first signs or symptoms. The clinical manifestations of the disease include rickets-like bone changes, bone demineralization, fragility fractures, reduced muscular strength, chest deformity, pulmonary hypoplasia, nephrolithiasis, nephrocalcinosis, and chondrocalcinosis. Treatment of HPP consists of enzyme replacement therapy. Before this therapy was approved, treatment was palliative and associated with high morbidity and mortality. Asfotase alfa has changed the prognosis of the disease by reducing bone deformity and improving bone mineralization, lung function, and muscle weakness, among other benefits. In adults, teriparatide and anti-sclerostin antibody have been used off-label in selected cases, demonstrating benefit in accelerating fracture healing and in concomitant treatment of osteoporosis. This review summarizes the main aspects of HPP and identifies the particularities of the disease in adult patients.

Dentoalveolar Alterations in an Adenine-Induced Chronic Kidney Disease Mouse Model.

Chronic kidney disease (CKD) is characterized by kidney damage and loss of renal function. CKD mineral and bone disorder (CKD-MBD) describes the dysregulation of mineral homeostasis, including hyperphosphatemia and elevated parathyroid hormone (PTH) secretion, skeletal abnormalities, and vascular calcification. CKD-MBD impacts the oral cavity, with effects including salivary gland dysfunction, enamel hypoplasia and damage, increased dentin formation, decreased pulp volume, pulp calcifications, and altered jaw bones, contributing to clinical manifestations of periodontal disease and tooth loss. Underlying mechanisms are not fully understood, and CKD mouse models commonly require invasive procedures with high rates of infection and mortality. We aimed to characterize the dentoalveolar effects of an adenine diet (AD)-induced CKD (AD-CKD) mouse model. Eight-week-old C57BL/6J mice were provided either a normal phosphorus diet control (CTR) or adenine and high-phosphorus diet CKD to induce kidney failure. Mice were euthanized at 15 weeks old, and mandibles were collected for micro-computed tomography and histology. CKD mice exhibited kidney failure, hyperphosphatemia, and hyperparathyroidism in association with porous cortical bone in femurs. CKD mice showed a 30% decrease in molar enamel volume compared to CTR mice. Enamel wear was associated with reduced ductal components, ectopic calcifications, and altered osteopontin (OPN) deposition in submandibular salivary glands of CKD mice. Molar cusps in CKD mice were flattened, exposing dentin. Molar dentin/cementum volume increased 7% in CKD mice and pulp volume decreased. Histology revealed excessive reactionary dentin and altered pulp-dentin extracellular matrix proteins, including increased OPN. Mandibular bone volume fraction decreased 12% and bone mineral density decreased 9% in CKD versus CTR mice. Alveolar bone in CKD mice exhibited increased tissue-nonspecific alkaline phosphatase localization, OPN deposition, and greater osteoclast numbers. AD-CKD recapitulated key aspects reported in CKD patients and revealed new insights into CKD-associated oral defects. This model has potential for studying mechanisms of dentoalveolar defects or therapeutic interventions. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Prenatal asfotase alfa-mediated enzyme replacement therapy restores delayed calcification in a severe infantile form of hypophosphatasia model mice

Hypophosphatasia (HPP) is a congenital disorder caused by mutations in the tissue-nonspecific alkaline phosphatase (TNALP) gene. The pathogenesis of HPP varies, ranging from severe cases in which there is total absence of fetal bone calcification, which leads to stillbirth, to relatively mild cases in which the effects are confined to the teeth, such as early loss of the primary teeth. In recent years, the establishment of enzyme supplementation as a treatment method has prolonged survival in patients; however, this approach does not provide sufficient improvement for failed calcification. Furthermore, the effects of enzyme replacement therapy on the jawbone and periodontal tissues have not yet been studied in detail. Therefore, in this study, we investigated the therapeutic effects of enzyme replacement therapy on jawbone hypocalcification in mice. Recombinant TNALP was administered to mothers before birth and newborns immediately after birth, and the effect of treatment was evaluated at 20 days of age. The treated HPP mice had improved mandible (mandibular length and bone quality) and tooth quality (root length of mandibular first molar, formation of cementum), as well as improved periodontal tissue structure (structure of periodontal ligament). Furthermore, prenatal treatment had an additional therapeutic effect on the degree of mandible and enamel calcification. These results suggest that enzyme replacement therapy is effective for the treatment of HPP, specifically in the maxillofacial region (including the teeth and mandible), and that early initiation of treatment may have additional beneficial therapeutic effects.

Promotion effect of FGF23 on osteopenia in congenital scoliosis through FGFr3/TNAP/OPN pathway.

Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23. We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined. DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown. Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP/OPN pathway.

Depot medroxyprogesterone acetate (DMPA)-associated early-onset osteoporotic fracture with ALPL gene mutation: Challenges and Limitations in the era of molecular diagnostics

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract #1412436: Nonclassical Presentation of Hypophosphatasia

Hypophosphatasia (HPP) is a rare genetic disorder characterized by impaired mineralization (calcification of bones and teeth). The signs and symptoms of HPP vary widely and can appear from birth to adulthood. We report a case of delayed bone fracture healing in the setting of hypophosphatasia. A 48-year-old male patient with a past medical history of vitamin D deficiency was seen in the outpatient clinic for low ALP levels. He had delayed wound healing involving the left clavicular and multiple rib fractures after a motor vehicle accident that occurred in 2012, which ultimately needed multiple surgeries requiring 1.5 years to fully heal/fuse. This involved placing and removing multiple plates and screws as well as bone grafts. He denied other fractures, skeletal deformities, or dental problems. He does have normal development and does not have short stature. He has a history of vitamin D deficiency and is on vitamin D 2000 units daily. He was found to have a family history remarkable with two sons with low ALP and vitamin D beginning in childhood. The older son was noted to have lost his teeth early at age 3-4 years, with no history of fractures. Musculoskeletal examination: no swelling noted, and no tenderness was noted but the patient did have a bone spur on the elbow, knee, and heel. He is able to ambulate without assistance and was found to have no other skeletal deformity. Laboratory findings: ALP 31 U/L (Normal 34-122). Bone-specific alkaline phosphatase: 5.6 ug/L (Normal 6.5-20.1). Vitamin D 25 OH level: 84 (on replacement therapy). Calcium: 9.2 mg/dl, phosphorus 3.5 mg/dl, iPTH levels 23 pg/ml. DXA scan: shows T values on the lumbar spine of -1.8, right hip of -1.4, and right neck of -1.9, consistent with osteopenia. We checked vitamin B6 level: 1087.7 nmol/L, consistent with hypophosphatasia. The patient then underwent genetic testing and is positive for ALPL gene mutation (heterozygous). Both his 2 sons are positive for ALPL gene mutation. We present a case of hypophosphatasia with nonclassical presentations, his mild symptoms likely from incomplete penetration. HPP causes pathogenic variants in ALPL gene. This gene provides instruction for making an enzyme called tissue non-specific alkaline phosphatase (TNSALP) which plays an essential role in the mineralization of the skeleton and teeth. Mutation in the ALP gene leads to the production of an abnormal version of TNSALP that cannot participate effectively in the mineralization process. There is currently no cure for hypophosphatasia, however human recombinant enzyme replacement therapy called asfotase alfa is now available.

Abstract #1377932: An Inherited Metabolic Disorder That Masquerades as Osteoporosis

Hypophosphatasia is a rare genetic disorder caused by loss-of-function mutations in the ALPL gene, which encodes tissue non-specific alkaline phosphatase. The enzyme defect results in the accumulation of substrates like inorganic pyrophosphate, thereby inhibiting mineralization of bones and teeth. Clinical features include premature loss of deciduous teeth, bowed legs, lower extremity stress fractures, bone pain and impaired mobility. Serum alkaline phosphatase (ALP) is a marker of bone turnover. Low ALP, high serum vitamin B6 and high urine phosphoethanolamine are diagnostic hallmarks of hypophosphatasia. A 54-year-old lady was referred to the endocrinology clinic for severe osteoporosis, characterized by an olecranon fracture and low bone mineral density (T score -3.0 at the left femoral neck). Bone disease risk factors included premature menopause at age 38 years and maternal history of hip fracture. There was no personal or family history of recurrent fractures, impaired mobility, or unexplained tooth loss. Despite treatment with ibandronate, zoledronic acid and teriparatide, her bone mineral density declined over the course of a decade. Physical examination revealed low BMI (18.07 kg/m2), normal gait and intact muscle strength. Lab testing disclosed low serum alkaline phosphatase (ALP) 18 units/L [reference 45 to 117 units/L], high serum vitamin B6 131.8 nmol/L [reference 20.0 to 125.0 nmol/L] and high urine phosphoethanolamine 104 nmol/mg Cr [reference < 48]. ALP results prior to bisphosphonate exposure were unavailable. Vitamin D status, thyroid function, parathyroid hormone, corrected serum calcium, ceruloplasmin and zinc levels were normal. Exome sequencing identified a pathogenic heterozygous nonsense ALPL gene mutation, c.1282C >T (p.Arg428X). She was counselled to avoid bisphosphonate therapy indefinitely. ALP measurement was advised for her blood relatives since hypophosphatasia is an autosomal recessive or dominant disorder. With asfotase alfa, the only FDA-approved therapy for juvenile-onset hypophosphatasia, there have been no further fractures to date, and her mobility remains unimpaired. Hypophosphatasia can be mistaken for osteoporosis since both conditions present with low bone mineral density on DEXA scan. Furthermore, bisphosphonate therapy and hypophosphatasia are mutually exclusive entities that can present with low ALP. Thus, bisphosphonate exposure may pose a diagnostic quandary when distinguishing between osteoporosis and hypophosphatasia. This case highlights the importance of assessing ALP when evaluating osteoporosis with atypical features such as age of onset under fifty years, remarkably low bone mineral density, fracture(s), and inadequate response to anti-osteoporosis agents. Moreover, if ALP is low, some index of suspicion for hypophosphatasia ought to be maintained, even if dental or lower extremity bone symptoms are absent. Genetic testing is confirmatory. Bisphosphonate therapy and parathyroid hormone analogs are not established treatment modalities for hypophosphatasia. Instead, enzyme replacement with asfotase alfa is recommended.

Development of gene therapy for hypophosphatasia

Hypophosphatasia (HPP) is a disease caused by an inborn defect in the production of alkaline phosphatase that affects bones and teeth. The six types are perinatal lethal form, perinatal benign form, infantile form, childhood form, adulthood form and odontohypophosphatasia. Treatments have historically been limited but, in 2015, an enzyme replacement therapy known as Asfotase Alfa was approved. This has improved life expectancy and quality of life but drawbacks such as requires 3-6 injections per week for life, injection pain and swelling at injection sites remain. Professor Koichi Miyake and his team at the Department of Gene Therapy at Nippon Medical School (NMS), Japan, are working to improve treatments for HPP. The researchers are developing novel therapeutic agents to potentially treat HPP by only single injection, one of which is ARU-2801. The hope is that this will not only prolong the life of HPP patients and improve their symptoms, but also improve their quality of life and will also improve the symptoms of patients with disease types that currently cannot be treated with enzyme replacement therapy. Miyake and the team have evaluated the efficacy and safety of ARU-2801 using non-human primates (NHPs). In their studies, ARU-2801 was able to maintain tissue nonspecific alkaline phosphatase (TNALP) expression in NHPs and mice for a long period, with no side effects. In future clinical trials the effectiveness of ARU-2801 will need to be confirmed in patients.

Exploring chromone sulfonamides and sulfonylhydrazones as highly selective ectonucleotidase inhibitors: Synthesis, biological evaluation and in silico study

Ectonucleotidases, a well-known superfamily of plasma membrane located metalloenzymes plays a central role in mediating the process of purinergic cell signaling. Major functions performed by these enzymes include the hydrolysis of extracellular nucleosides and nucleotides which are considered as important cell-signaling molecules. Any (patho)-physiologically induced disruption in this purinergic cell signaling leads to several disorders, hence these enzymes are important drug targets for therapeutic purposes. Among the major challenges faced in the design of inhibitors of ectonucleotidases, an important one is the lack of selective inhibitors. Access to highly selective inhibitors via a facile synthetic route will not only be beneficial therapeutically, but will also lead to an increase in our understanding of intricate interplay between members of ectonucleotidase enzymes in relation to their selective activation and/or inhibition in different cells and tissues. Herein we describe synthesis of highly selective inhibitors of human intestinal alkaline phosphatase (h-IAP) and human tissue non-specific alkaline phosphatase (h-TNAP), containing chromone sulfonamide and sulfonylhydrazone scaffolds. Compound 1c exhibited highest (and most selective) h-IAP inhibition activity (h-IAP IC50 = 0.51 ± 0.20 µM; h-TNAP = 36.5%) and compound 3k showed highest activity and selective inhibition against h-TNAP (h-TNAP IC50 = 1.41 ± 0.10 µM; h-IAP = 43.1%). These compounds were also evaluated against another member of ectonucleotidase family, that is rat and human ecto-5′-nucleotidase (r-e5′NT and h-e5′NT). Some of the compounds exhibited excellent inhibitory activity against ecto-5′-nucleotidase. Compound 2 g exhibited highest inhibition against h-e5′NT (IC50 = 0.18 ± 0.02 µM). To rationalize the interactions with the binding site, molecular docking studies were carried out.

Disulfide Bonds Are Not Necessary for Intrinsic TNSALP Activity.

Recent developments in single-molecule enzymology (SME) have allowed for the observation of subpopulations present in enzyme ensembles. Tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric monophosphate esterase central to bone metabolism, has become a model enzyme for SME studies. TNSALP contains two internal disulfide bonds that are critical for its effective dimerization; mutations in its disulfide bonding framework have been reported in patients with hypophosphatasia, a rare disease characterized by impaired bone and tooth mineralization. In this paper, we present the kinetics of these mutants and show that these disulfide bonds are not crucial for TNSALP enzymatic function. This surprising result reveals that the enzyme's active conformation does not rely on its disulfide bonds. We posit that the signs and symptoms seen in hypophosphatasia are likely not primarily due to impaired enzyme function, but rather decreased enzyme expression and trafficking.

Exploration of Novel Genetic Evidence and Clinical Significance Into Hemifacial Microsomia Pathogenesis

The authors browsed through past genetic findings in hemifacial microsomia along with our previously identified mutations in ITGB4 and PDE4DIP from whole genome sequencing of hemifacial microsomia patients. Wondering whether these genes influence mandibular bone modeling by regulation on osteogenesis, the authors approached mechanisms of hemifacial microsomia through this investigation into gene knockdown effects in vitro. MC3T3E1 cells were divided into 5 groups: the negative control group without osteogenesis induction or siRNA, the positive control group with only osteogenesis induction, and 3 gene silenced groups with both osteogenesis induction and siRNA. Validation of transfection was through fluorescence microscopy and quantitative real-time Polymerase chain reaction on knockdown efficiency. Changes in expression levels of the 3 genes during osteogenesis and impact of Itgb4 and Pde4dip knockdown on osteogenesis were examined by quantitative real-time Polymerase chain reaction, alkaline phosphatase, and alizarin red staining. Elevation of osteogenic genes Alpl, Col1a1, Bglap, Spp1, and Runx2 verified successful osteogenesis. Both genes were upregulated under osteogenic induction, while they had different trends over time. Intracellular fluorophores under microscope validated successful transfection and si-m-Itgb4_003, si-m-Pde4dip_002 had satisfactory knockdown effects. During osteogenesis, Pde4dip knockdown enhanced Spp1 expression (1.95±0.13 folds, P =0.045). The authors speculated that these genes may have different involvements in osteogenesis. Stimulated expression of Spp1 by Pde4dip knockdown may suggest that Pde4dip inhibits osteogenesis.

Alkaline phosphatase in clinical practice in childhood: Focus on rickets.

Serum alkaline phosphatase (ALP) and its isoenzymes reflect bone metabolism: ALP increases the ratio of inorganic phosphate to pyrophosphate systemically and facilitates mineralization as well as reduces extracellular pyrophosphate concentration, an inhibitor of mineral formation. On the contrary, low ALP activity is associated with reduction of bone turnover. ALP includes four isoenzymes depending on the site of tissue expression: intestinal ALP, placental ALP, germ cell ALP and tissue nonspecific ALP or liver/bone/kidney ALP. The bone isoenzyme (B-ALP) is involved in bone calcification and is a marker of bone turnover as a result of osteoblastic activity. ALP and its isoenzymes are crucial in the diagnostic process of all the forms of rickets.The most common cause of rickets is vitamin D nutritional deficiency. The aim of this review is to update on the role played by ALP serum concentrations as a relevant marker in thediagnosis and treatment of rickets. Indeed, the diagnosis of rickets is based on its clinical, radiological and laboratory characteristics. An elevated ALP level is one of the markers for the diagnosis of rickets in children, though it is also associated with bone formation process. ALP is also useful for the differentiation between rickets and other disorders that can mimic rickets because of their clinical and laboratory characteristics, and, together with other biochemical markers, is crucial for the differential diagnosis of the different forms of rickets. Age, severity and duration of rickets may also modulate ALP elevation. Finally, ALP measurements are useful in clinical and therapeutic follow-up.

Abstract 19: Loss Of Endothelial Tissue-nonspecific Alkaline Phosphatase Modifies Sensorimotor Deficits In Chronic Ischemic Stroke

Tissue nonspecific alkaline phosphatase (TNAP) is an ectoenzyme that is known for its anti-inflammatory properties in the immune system. Although TNAP is present in nearly every cell, it is highly abundant in brain endothelial cells (BEC). However, the role of TNAP in the BECs and other cells in the neurovascular unit is poorly understood. Our laboratory has previously identified a role for BEC TNAP in vascular integrity. While the loss of cortical TNAP activity has been observed in ischemic stroke, the functional outcomes that result from the loss of the enzyme are less clear. The objective of this study is to determine how TNAP affects post-stroke functional outcomes. We hypothesized that the loss of endothelial TNAP worsens post-stroke sensorimotor deficits. To test this, we generated mice with a conditional deletion of Alpl , the gene that codes for TNAP, in endothelial cells (VE-cKO mice) and compared the results to their littermate controls ( Alpl fl/fl ). Male and female six month-old mice (n=40) received a photothrombotic ischemic stroke (PTS) or sham surgery followed by daily neurological assessment (Clark’s test) and comprehensive sensorimotor testing, i.e. corner test, rotarod, open field, and gait analysis (DigiGait), over a seven-day period. Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p&lt;0.0001). Daily neurological scoring showed higher (worse) scores in VEcKO PTS-mice compared to littermate controls (p=0.004). Quantification of total spontaneous movement via open field testing showed a significant interaction among stroke, genotype, and time (p=0.014). Gait analysis showed that the post-stroke stance/swing ratio, a measure of stability, differed between the left and right hind paws in VEcKO mice compared to controls: right hind paw- genotype x stroke interaction (p=0.041), and left hind paw- genotype x surgery x sex interaction (p=0.005). Significant interactions between surgery and sex were also observed in the right fore paw for stride (p=0.02), stride frequency (p=0.00026), and stride length (p=0.02). These results demonstrate a novel role for endothelial TNAP in functional recovery post-stroke.

A rare case of adult hypophosphatasia masked with primary hyperparathyroidism

Abstract Introduction Hypophosphatasia (HPP) is a rare inherited disorder of bone and mineral metabolism due to mutations in the Alkaline Phosphatase-Liver (ALPL) gene encoding the tissue nonspecific alkaline phosphatase (TNSALP). Clinical features of HPP in adults include early loss of teeth, muscle weakness, musculoskeletal pain, osteoporosis and fragility fractures with delayed healing. The most typical biochemical manifestation of the disease is persistently low level of alkaline phosphatase (ALP). Primary hyperparathyroidism (PHPT) is a relatively common endocrine disorder leading to increase in bone turnover markers including ALP. Coexistence of HPP and PHPT is a very rare condition. Herein we present an adult HPP patient who presented with primary hyperparathyroidism and normal serum ALP levels, which decreased significantly after parathyroidectomy. Clinical Case A 33-year-old female with a past medical history of Hashimoto's thyroiditis and acne rosacea admitted to our outpatient clinic because of gradually increasing bone pain, fatigue and weakness. She had early teeth loss and dental problems, but she denied history of fractures and kidney stones. Her family history was remarkable for nephrolithiasis in both parents. On physical examination the patient had facial rash and telangiectasia. She also had teeth loss and many dental cavities. Her vital signs were within normal limits. In biochemical investigations creatinine was 0.45 mg/dL (0.7–1.4), ALP 81 U/L (35–105), PTH 531 pg/mL (15–65), calcium 10.84 mg/dL (8.5–10.5), albumin 4.9 g/dL (3.2–5.5), phosphorus 2.25 mg/dL (2.7–4.5), Vitamin D 5 ng/mL and urine calcium excretion 613 mg/day. Laboratory results were compatible with normocalcemic hyperparathyroidism and severe vitamin D deficiency. Bone mineral density of the lumbar spine and the femoral neck revealed osteopenia and 1/3 distal radius revealed osteoporosis. There was no evidence of nephrolithiasis and nephrocalcinosis on abdominal ultrasound. Neck ultrasound and CT scan and Tc-99m MIBI scanning of the patient revealed a parathyroid adenoma in the posteroinferior of thyroid gland's left lobe. A successful parathyroidectomy was performed. During follow-up the patient's serum ALP levels decreased significantly to 29 U/L with a repeat being 26 U/L. Bone-specific ALP was 4.2 μg/L (3–14 μg/L). Urine phosphoethanolamine was elevated to 68.6 μmol/g.Cr (0–48), which supported the diagnosis of HPP. Genetic analysis was performed and heterozygous p.N190del (c.568_570delAAC/c.566_568delACA) mutation was detected. This mutation was previously reported as pathogenic and has been associated with adult hypophosphatasia. Conclusion Although persistently low level of ALP is characteristic of HPP, disorders that increase bone turnover may result in an increase in serum ALP levels, which may mask underlying HPP. Acknowledgements We are thankful to Alexion Pharmaceuticals, Inc for providing fund for ALP substrates and genetic analysis of the patient.

Cirsium Setidens Water Extracts Containing Linarin Block Estrogen Deprivation-Induced Bone Loss in Mice.

Osteoporosis is evident in postmenopausal women and is an osteolytic disease characterized by bone loss that further increases the susceptibility to bone fractures and frailty. The use of complementary therapies to alleviate postmenopausal osteoporosis is fairly widespread among women. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. This study aimed to determine whether Cirsium setidens water extracts (CSEs), the component linarin, and its aglycone acacetin blocked ovariectomy (OVX)-induced bone loss. This study employed OVX C57BL/6 female mice as a model for postmenopausal osteoporosis. CSEs, acacetin, or linarin was orally administrated to OVX mice at a dose of 20 mg/kg for 8 weeks. Surgical estrogen loss in mice for 8 weeks reduced bone mineral density (BMD) of mouse femur and serum 17β-estradiol level and enhanced the serum receptor activator of NF-κB ligand/osteoprotegerin ratio with uterine atrophy. CSEs and linarin reversed such adverse effects and enhanced femoral BMD in OVX mice. Oral administration of CSEs and linarin attenuated tartrate-resistant acid phosphate activity and the induction of αvβ3 integrins and proton suppliers in resorption lacunae in femoral bone tissue of OVX mice. In addition, CSEs and linarin curtailed the bone levels of cathepsin K and matrix metalloproteinase-9 responsible for osteoclastic bone resorption. On the other hand, CSEs and linarin enhanced the formation of trabecular bones in estrogen-deficient femur with increased induction of osteocalcin and osteopontin. Further, treatment with CSEs and linarin enhanced the collagen formation-responsive propeptide levels in the circulation along with the increase in the tissue non-specific alkaline phosphatase level in bone exposed to OVX. Supplementing CSEs, acacetin, or linarin to OVX mice elevated the formation of collagen fibers in OVX trabecular bone, evidenced using Picrosirius red staining. Accordingly, CSEs and linarin were effective in retarding osteoclastic bone resorption and promoting osteoblastic bone matrix mineralization under OVX conditions. Therefore, linarin, which is abundant in CSEs, may be a natural compound for targeting postmenopausal osteoporosis and pathological osteoresorptive disorders.

Synthesis, kinetic studies and in-silico investigations of novel quinolinyl-iminothiazolines as alkaline phosphatase inhibitors

Deposition of hydroxyapatite (HA) or alkaline phosphate crystals on soft tissues causes the pathological calcification diseases comprising of end-stage osteoarthritis (OA), ankylosing spondylitis (AS), medial artery calcification and tumour calcification. The pathological calcification is symbolised by increased concentration of tissue non-specific alkaline phosphatase (TNAP). An efficient therapeutic strategy to eradicate these diseases is required, and for this the alkaline phosphatase inhibitors can play a potential role. In this context a series of novel quinolinyl iminothiazolines was synthesised and evaluated for alkaline phosphatase inhibition potential. All the compounds were subjected to DFT studies where N-benzamide quinolinyl iminothiazoline (6g), N-dichlorobenzamide quinolinyl iminothiazoline (6i) and N-nitrobenzamide quinolinyl iminothiazoline (6j) were found as the most reactive compounds. Then during the in-vitro testing, the compound N-benzamide quinolinyl iminothiazoline (6g) exhibited the maximum alkaline phosphatase inhibitory effect (IC50 = 0.337 ± 0.015 µM) as compared to other analogues and standard KH2PO4 (IC50 = 5.245 ± 0.477 µM). The results were supported by the molecular docking studies, molecular dynamics simulations and kinetic analysis which also revealed the inhibitory potential of compound N-benzamide quinolinyl iminothiazoline (6g) against alkaline phosphatase. This compound can be act as lead molecule for the synthesis of more effective inhibitors and can be suggested to test at the molecular level.

A unique case of childhood hypophosphatasia caused by a novel heterozygous 51-bp in-frame deletion in the &lt;i&gt;ALPL&lt;/i&gt; gene

Hypophosphatasia (HPP) is caused by inactivating variants of the ALPL gene, which encodes tissue non-specific alkaline phosphatase (TNSALP). Among the six subtypes of HPP, childhood HPP presents after 6 months and before 18 yr of age, and is inherited in both autosomal dominant and autosomal recessive manners. Patients with childhood HPP have variable symptoms, including rickets-like bone changes, low bone mineral density (BMD), short stature, muscle weakness, craniosynostosis, and premature loss of deciduous teeth. Here, we describe a 7-yr-old boy with childhood HPP who showed short stature, impaired ossification of the carpal bones, and low BMD. Genetic testing identified a novel heterozygous 51-bp in-frame deletion in the ALPL gene (c.1482_1532del51), leading to the lack of 17 amino acids between Gly495 and Leu511 (p.Gly495_Leu511del). In vitro transfection experiments revealed the loss of enzymatic activity and the dominant-negative effect of the TNSALP[p.Gly495_Leu511del] variant; thus, the patient was diagnosed as having autosomal dominant HPP. The TNSALP[p.Gly495_Leu511del] variant was localized to the plasma membrane as was the wild-type TNSALP (TNSALP[WT]): however, co-immunoprecipitation experiments suggested a reduced dimerization between TNSALP[p.Gly495_Leu511del] and TNSALP[WT]. This case expands the variable clinical manifestation of childhood HPP and sheds light on the molecular bases underlying the dominant-negative effects of some TNSALP variants.

Humoral and cellular factors inhibit phosphate-induced vascular calcification during the growth period.

Hyperphosphatemia is an independent and non-classical risk factor of cardiovascular disease and mortality in patients with chronic kidney disease (CKD). Increased levels of extracellular inorganic phosphate (Pi) are known to directly induce vascular calcification, but the detailed underlying mechanism has not been clarified. Although serum Pi levels during the growth period are as high as those observed in hyperphosphatemia in adult CKD, vascular calcification does not usually occur during growth. Here, we have examined whether the defence system against Pi-induced vascular calcification can exist during the growth period using mice model. We found that calcification propensity of young serum (aged 3 weeks) was significantly lower than that of adult serum (10 months), possibly due to high fetuin-A levels. In addition, when the aorta was cultured in high Pi medium in vitro, obvious calcification was observed in the adult aorta but not in the young aorta. Furthermore, culture in high Pi medium increased the mRNA level of tissue-nonspecific alkaline phosphatase (TNAP), which degrades pyrophosphate, only in the adult aorta. Collectively, our findings indicate that the aorta in growing mouse may be resistant to Pi-induced vascular calcification via a mechanism in which high serum fetuin-A levels and suppressed TNAP expression.