Prevalence of Low Serum Alkaline Phosphatase and Hypophosphatasia in Adult Patients with Atypical Femur Fractures.

Hypophosphatasia (HPP) is a rare genetic disorder characterized by low serum alkaline phosphatase (ALP), its manifestations may include atypical femoral fractures (AFF). However, the prevalence of low serum ALP and HPP in patients with AFF remains unknown. We retrospectively analyzed ALP levels and clinical manifestations compatible with HPP in 72 adult patients with confirmed AFF by chart review. ALP values were compared with those of a control group of patients with prior proximal femoral fracture during antiresorptive treatment (n = 20). Among the AFF patients, 18 (25%) had at least one serum ALP value ≤ 40 IU/L, although in all but one case, at least one ALP value > 40 IU/L was also detected at another time point. Most low ALP values were associated with antiresorptive treatment (P = 0.049) and lowest levels of ALP did not differ between the AFF and the control groups (P = 0.129). However, low ALP values among AFF patients were associated with a higher rate of bilateral AFF (50% vs 22%, P = 0.025), metatarsal fracture (33% vs 7%, P = 0.006), and with trends for more frequent use of glucocorticoid (22% vs 8%, P = 0.089) and proton pump inhibitor (61% vs 44%, P = 0.220). In one AFF patient with low ALP and clinical suspicion of HPP, a rare pathogenic heterozygous variant of the ALPL gene was identified. In conclusion, low ALP values are common among subjects with AFF and mainly related to concomitant antiresorptive medication. Hence, low serum ALP has low specificity for HPP among AFF patients.

Cross-sectional analysis: clinical presentation of children with persistently low ALP levels.

OBJECTIVES: Low activity of serum alkaline phosphatase (ALP) is a hallmark of hypophosphatasia (HPP), but low readings of ALP are not always recognized in clinical routine. Understanding the clinical presentations associated with low ALP may contribute to a timelier diagnosis of HPP. METHODS: Data from paediatric patients with low ALP, excluding patients in intensive care and with oncological/haematological disorders, were analysed. Most recent ALP values, previous diagnoses, medication and relevant symptoms were extracted from patient records at nine specialised centres and analysed descriptively. A relationship between body height and ALP values was scrutinised by linear regression. RESULTS: Of 370 children, 15 (4.1%) had a diagnosis of HPP. In the subgroup without a diagnosis of HPP, 241 (67.9%) out of 355 patients had one or more medical conditions known to be associated with low serum ALP. Of those, hypothyroidism, malnutrition and steroid administration were most frequent. Characteristic symptoms, particularly, short stature, muscle weakness and delay of motor development were more frequent and ALP values were lower in patients with documented HPP diagnosis compared to patients without diagnosis of HPP (Ø z-scores: -2.52) (interquartile range [IQR] = 0.20) vs. -1.96 (IQR = 0.87). A weak positive linear relationship between z-scores of ALP and body height was identified (p<0.001). CONCLUSIONS: This analysis of paediatric patient records elucidates a wide range of disorders associated with low ALP activity. In case of additional specific symptoms, HPP should always be considered as a differential diagnosis.

Hypophosphatasia: A Unique Disorder of Bone Mineralization.

Hypophosphatasia (HPP) is a rare genetic disease characterized by a decrease in the activity of tissue non-specific alkaline phosphatase (TNSALP). TNSALP is encoded by the ALPL gene, which is abundantly expressed in the skeleton, liver, kidney, and developing teeth. HPP exhibits high clinical variability largely due to the high allelic heterogeneity of the ALPL gene. HPP is characterized by multisystemic complications, although the most common clinical manifestations are those that occur in the skeleton, muscles, and teeth. These complications are mainly due to the accumulation of inorganic pyrophosphate (PPi) and pyridoxal-5'-phosphate (PLP). It has been observed that the prevalence of mild forms of the disease is more than 40 times the prevalence of severe forms. Patients with HPP present at least one mutation in the ALPL gene. However, it is known that there are other causes that lead to decreased alkaline phosphatase (ALP) levels without mutations in the ALPL gene. Although the phenotype can be correlated with the genotype in HPP, the prediction of the phenotype from the genotype cannot be made with complete certainty. The availability of a specific enzyme replacement therapy for HPP undoubtedly represents an advance in therapeutic strategy, especially in severe forms of the disease in pediatric patients.

Hypophosphatasia: is it an underdiagnosed disease even by expert physicians?

INTRODUCTION: Hypophosphatasia (HPP) is caused by mutations in the ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (ALP). Clinical manifestations range from extreme life-threatening lethal forms to no signs or symptoms at all. MATERIALS AND METHODS: Consecutive 30,000 outpatients and inpatients with ALP data were screened retrospectively, out of which 1000 patients were found to have low levels of ALP more than once. Then, patients were evaluated for the symptoms and signs of HPP with further biochemical and genetic analyses. RESULTS: Thirty-seven patients who had severe musculoskeletal pain, recurrent fractures, and tooth anomalies were then screened with substrate and DNA sequencing analyses for HPP. It was determined that eight patients had variants in the ALPL gene. A total of eight different ALPL variants were identified in eight patients. The variants, namely c.244G > C (p.Gly82Arg), c.1444C > T (p.His482Tyr), c.1487A > G (p.Asn493Ser), and c.675_676insCA (p.Met226GlnfsTer52), had not been previously reported. DISCUSSION: Considering the wide spectrum of clinical signs and symptoms, HPP should be among the differential lists of bone, muscle, and tooth abnormalities at any age.

Tissue-Nonspecific Alkaline Phosphatase-A Gatekeeper of Physiological Conditions in Health and a Modulator of Biological Environments in Disease.

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5'-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the ALPL gene and consequently altered TNAP activity are suffering from the rare metabolic disease hypophosphatasia (HPP). This systemic disease is mainly characterized by impaired bone and dental mineralization but may also be accompanied by neurological symptoms, like anxiety disorders, seizures, and depression. HPP characteristically affects all ages and shows a wide range of clinical symptoms and disease severity, which results in the classification into different clinical subtypes. This review describes the molecular function of TNAP during the mineralization of bones and teeth, further discusses the current knowledge on the enzyme's role in the nervous system and in sensory perception. An additional focus is set on the molecular role of TNAP in health and on functional observations reported in common laboratory vertebrate disease models, like rodents and zebrafish.

Intrafamilial phenotypic distinction of hypophosphatasia with identical tissue nonspecific alkaline phosphatase gene mutation: a family report.

Hypophosphatasia (HPP) is caused by mutations in the tissue nonspecific alkaline phosphatase (TNSALP) gene in an autosomal recessive or dominant manner and characterized by defective mineralization of bone and low serum ALP levels. In this report, we present a family with HPP mother (case 1) and HPP child (case 2) who have identical TNSALP gene mutation (c.1015G>A p.Gly339Arg heterozygous mutation) but distinct clinical phenotypes. Whereas case 1 appeared to be asymptomatic despite extremely low levels of serum ALP, case 2 had several HPP-related symptoms, such as tooth loss, fractures, short stature, with slightly decreased ALP levels. Upon the diagnosis of HPP, case 1 discontinued denosumab, which was used to treat her rheumatoid arthritis, concerning the risk of atypical femoral fractures. The clinical course of this family was suggestive in a genotype-phenotype imbalance in HPP, the underdiagnosis of HPP in adults, and the risk of atypical femoral fractures using bone resorption inhibitors.

Alkaline phosphatase: Structure, expression and its function in bone mineralization.

Alkaline phosphatase (ALP) is highly expressed in the cells of mineralized tissue and plays a critical function in the formation of hard tissue. The existing status of this critical enzyme should be reviewed periodically. ALP increases inorganic phosphate local rates and facilitates mineralization as well as reduces the extracellular pyrophosphate concentration, an inhibitor of mineral formation. Mineralization is the production, inside matrix vesicles, of hydroxyapatite crystals that bud from the outermembrane of hypertrophic osteoblasts and chondrocytes. The expansion of hydroxyapatite formsinto the extracellular matrix and its accumulation between collagen fibrils is observed. Among various isoforms, the tissue-nonspecific isozyme of ALP (TNAP) is strongly expressed in bone, liver and kidney and plays a key function in the calcification of bones. TNAP hydrolyzes pyrophosphate and supplies inorganic phosphate to enhance mineralization. The biochemical substrates of TNAP are believed to be inorganic pyrophosphate and pyridoxal phosphate. These substrates concentrate in TNAP deficient condition which results in hypophosphatasia. The increased level of ALP expression and development in this environment would undoubtedly provide new and essential information about the fundamental molecular mechanisms of bone formation, offer therapeutic possibilities for the management of bone-related diseases.

Case Report: Efficacy of Reduced Doses of Asfotase Alfa Replacement Therapy in an Infant With Hypophosphatasia Who Lacked Severe Clinical Symptoms.

Background: Hypophosphatasia is a rare bone disease characterized by impaired bone mineralization and low alkaline phosphatase activity. Here, we describe the course of bone-targeted enzyme replacement therapy with asfotase alpha for a female infant patient with hypophosphatasia who lacked apparent severe clinical symptoms. Case presentation: The patient exhibited low serum alkaline phosphatase (60 U/L; age-matched reference range, 520-1,580) in a routine laboratory test at birth. Further examinations revealed skeletal demineralization and rachitic changes, as well as elevated levels of serum calcium (2.80 mmol/L; reference range, 2.25-2.75 mmol/L) and ionic phosphate (3.17 mmol/L; reference range, 1.62-2.48 mmol/L), which are typical features in patients with hypophosphatasia. Sequencing analysis of the tissue-nonspecific alkaline phosphatase (TNSALP) gene identified two pathogenic mutations: c.406C>T, p.Arg136Cys and c.979T>C, p.Phe327Leu. Thus, the patient was diagnosed with hypophosphatasia. At the age of 37 days, she began enzyme replacement therapy using asfotase alpha at the standard dose of 6 mg/kg/week. Initial therapy from the age of 37 days to the age of 58 days substantially improved rickets signs in the patient; it also provided immediate normalization of serum calcium and ionic phosphate levels. However, serum ionic phosphate returned to a high level (2.72 mmol/L), which was presumed to be a side effect of asfotase alpha. Thus, the patient's asfotase alfa treatment was reduced to 2 mg/kg/week, which allowed her to maintain normal or near normal skeletal features thereafter, along with lowered serum ionic phosphate levels. Because the patient exhibited slight distal metaphyseal demineralization in the knee at the age of 2 years and 6 months, her asfotase alfa treatment was increased to 2.4 mg/kg/week. No signs of deterioration in bone mineralization were observed thereafter. At the age of 3 years, the patient's motor and psychological development both appeared normal, compared with children of similar age. Conclusion: This is the first report in which reduced doses of asfotase alfa were administered to an infant patient with hypophosphatasia who lacked apparent severe clinical symptoms. The results demonstrate the potential feasibility of a tailored therapeutic option based on clinical severity in patients with hypophosphatasia.

Tissue non-specific alkaline phosphatase activity and mineralization capacity of bi-allelic mutations from severe perinatal and asymptomatic hypophosphatasia phenotypes: Results from an in vitro mutagenesis model.

BACKGROUND: Hypophosphatasia (HPP) is an inherited metabolic bone disease characterized by reduced mineralization due to mutations in the tissue non-specific alkaline phosphatase (ALPL) gene. HPP is clinically variable with extensive allelic heterogeneity in the ALPL gene. We report the findings of in vitro functional studies following site-directed mutagenesis in bi-allelic mutations causing extreme clinical phenotypes; severe perinatal and asymptomatic HPP. AIMS: Elucidate genotype-phenotype correlation using in vitro functional studies and 3 dimensional (3D) ALP modelling. METHODS: Clinical, biochemical and radiological features were recorded in two children with extreme HPP phenotypes: Subject 1 (S1): Perinatal HPP with compound heterozygous mutations (c.110T>C; c.532T>C); Subject 2 (S2): asymptomatic with homozygous missense mutation (c.715G>T). Plasmids created for mutants 1 c.110T>C (L37P), 2 c.532T>C (Y178H) and 3 c.715G>T (D239Y) using in vitro mutagenesis were transfected into human osteosarcoma (U2OS) cells and compared to wildtype (WT) and mock cDNA. ALP activity was measured using enzyme kinetics with p-nitrophenylphosphate. Mineral deposition was evaluated photometrically with Alizarin Red S staining after culture with mineralization medium. Western blot analysis was performed to identify the mature type protein expression (80 kDa). Mutations were located on a 3D ALP model. Co-transfection was performed to identify dominant negative effect of the mutants. RESULTS: Phenotype: S1, had typical perinatal HPP phenotype at birth; extremely under-mineralized bones and pulmonary hypoplasia. S2, diagnosed incidentally by laboratory tests at 4 years, had normal growth, development, dentition and radiology. All S2's siblings (3 homozygous, 1 heterozygous) were asymptomatic. All subjects had typical biochemical features of HPP (low ALP, high serum pyridoxal-5'-phosphate), except the heterozygous sibling (normal ALP). Functional assay: Mutants 1 and 2 demonstrated negligible ALP activity and mineralization was 7.9% and 9.3% of WT, respectively. Mutant 3 demonstrated about 50% ALP activity and 15.5% mineralization of WT. On Western blot analysis, mutants 1 and 2 were detected as faint bands indicating reduced expression and mutant 3 was expressed as mature form protein with 50% of WT expression. Mutant 1 was located near the Glycosylphosphatidylinositol anchor, 2 at the core structure of the ALP protein and 3 at the periphery of the protein structure. Co-transfection did not reveal a dominant negative effect in any of the mutants. CONCLUSION: Our findings expand the current knowledge of functional effect of individual mutations and the importance of their location in the ALP structure.

A Case of the Perinatal Form Hypophosphatasia Caused by a Novel Large Duplication of the ALPL Gene and Report of One Year Follow-up with Enzyme Replacement Therapy

Hypophosphatasia (HPP) is a rare disease caused by mutations in the ALPL gene encoding tissue-non-specific isoenzyme of alkaline phosphatase (TNSALP). Duplications of the ALPL gene account for fewer than 1% of the mutations causing HPP. It has been shown that asfotase alfa enzyme replacement treatment (ERT) mineralizes the skeleton and improves respiratory function and survival in severe forms of HPP. Our patient was a newborn infant evaluated for respiratory failure and generalized hypotonia after birth. Diagnosis of HPP was based on low-serum ALP activity, high concentrations of substrates of the TNSALP and radiologic findings. On day 21 after birth, ERT using asfotase alfa (2 mg/kg three times per week, subcutaneous injection) was started. His respiratory support was gradually reduced and skeletal mineralization improved during treatment. We were able to discharge the patient when he was seven months old. No mutation was detected in the ALPL gene by all exon sequencing, and additional analysis was done by quantitative polymerase chain reaction (qPCR). As a result, a novel homozygote duplication encompassing exons 2 to 6 was detected. Early diagnosis and rapid intervention with ERT is life-saving in the severe form of HPP. qPCR can detect duplications if a mutation cannot be detected by sequence analysis in these patients.

Childhood hypophosphatasia: to treat or not to treat.

BACKGROUND: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from dysfunction of the tissue non-specific alkaline phosphatase enzyme. Its manifestations are extremely variable, ranging from early lethality to disease limited to the dentition. The disease is life-threatening when manifesting within the first six months of life, excepting the extremely rare benign perinatal hypophosphatasia. Childhood hypophosphatasia, defined as onset of symptoms between six months and eighteen years, can manifest as rickets, pain, decreased mobility, deficits of growth, and fractures. Historical treatment has generally involved a combination of dietary and rehabilitative interventions. MAIN DOCUMENT: Asfotase alfa (Strensiq™), is a first-in-class bone-targeted recombinant tissue nonspecific alkaline phosphatase which has shown significant improvements in morbidity and mortality in patients with perinatal and infantile hypophosphatasia. Subsequent research has also shown improvements in morbidity for patients with childhood hypophosphatasia as measured by improvement in rickets, growth, strength, mobility, and quality of life. This enzyme replacement therapy has generally been well-tolerated, with most adverse reactions being mild-to-moderate in nature. The author shares their approach to decisions on commencement of ERT based from experience of managing approximately fifteen patients across the age spectrum. This approach focuses on assessing the severity of five key manifestations of childhood HPP: decreased mobility, pain, rickets, deficits of growth, and fractures.

Abnormal bone turnover in individuals with low serum alkaline phosphatase.

The clinical spectrum of hypophosphatasia (HPP) is broad and variable within families. Along severe infantile forms, adult forms with mild manifestations may be incidentally discovered by the presence of low alkaline phosphatase (ALP) activity in serum. However, it is still unclear whether individuals with persistently low levels of ALP, in the absence of overt manifestations of HPP, have subclinical abnormalities of bone remodeling or bone mass. The aim of this study was to obtain a better understanding of the skeletal phenotype of adults with low ALP by analyzing bone mineral density (BMD), bone microarchitecture (trabecular bone score, TBS), and bone turnover markers (P1NP and ß-crosslaps). We studied 42 individuals with persistently low serum ALP. They showed lower levels of P1NP (31.4 ± 13.7 versus 48.9 ± 24.4 ng/ml; p = 0.0002) and ß-crosslaps (0.21 ± 0.17 versus 0.34 ± 0.22 ng/ml, p = 0.0015) than individuals in the control group. There were no significant differences in BMD, bone mineral content, or TBS. These data suggest that individuals with hypophosphatasemia have an overall reduction of bone turnover, even in the absence of overt manifestations of HPP or low BMD. We evaluated bone mineral density (BMD), bone microarchitecture, and bone turnover markers in patients with low serum levels of alkaline phosphatase. Our results show that these patients have low bone remodeling even in the absence of BMD abnormalities, thus supporting the recommendation of avoiding antiresorptives such as bisphosphonates in these subjects.

A homozygous intronic branch-point deletion in the ALPL gene causes infantile hypophosphatasia.

Hypophosphatasia (HPP) is a multi-systemic inborn disease with an extraordinary spectrum of severity, ranging from the absence of mineralization to high lethality and it involves different organs including bone, muscle, kidney, lung, gastrointestinal tract and the nervous system. The disease is characterized by low levels of serum alkaline phosphatase, caused by loss-of-function mutations within the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase TNAP. Here we present the functional characterization of a gene mutation, detected in intron 7 of the ALPL gene of a boy with infantile HPP in whom routine sequencing of the coding region failed to detect any mutation. The homozygous c.793del-14_33 mutation results in the loss of the branch-point motif, relevant for correct ALPL pre-mRNA splicing. The main transcript skips exon 8 and codes for a C-terminally truncated TNAP protein of 275 amino acids, which was detected in peripheral blood mononuclear cells and serum from the patient. The functional characterization of recombinant TNAP275 revealed no enzymatic activity nor any dominant-negative effect, relevant for the heterozygous parents. Nevertheless correct pre-mRNA splicing can take place without the branch-point sequence to a limited extend, as concluded from the ALPL cDNA, obtained from patient's PBMC, and from the low serum AP activity. These data reaffirm that in clear cut clinical cases, where conventional sequencing including the coding sequence and direct exon-intron-boundaries fails to detect mutations, deeper analyses of regulatory important motifs like branch-point sequences are required to establish a genetic diagnosis.

Molecular defect of tissue-nonspecific alkaline phosphatase bearing a substitution at position 426 associated with hypophosphatasia.

Mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP) cause hypophosphatasia (HPP), a genetic disorder characterized by deficiency of serum ALP and hypomineralization of bone and teeth. Three missense mutations for glycine 426 (by standard nomenclature) of TNSALP have been reported: cysteine (p.G426C), serine (p.G426S), and aspartate (p.G426D). We expressed TNSALP mutants carrying each missense mutation in mammalian cells. All three TNSALP mutants appeared on the cell surface like the wild-type (WT) TNSALP, although the cells expressing each TNSALP mutant exhibited markedly reduced ALP activity. TNSALP (WT) was mainly present as a 140 kDa catalytically active dimeric form, whereas ~80 kDa monomers were the predominant molecular species in the cells expressing TNSALP (p.G426D) or TNSALP (p.G426S), suggesting that aspartate or serine at position 426 may hamper the subunit assembly essential for the enzymatic function of TNSALP. Alternatively, the subunits of TNSALP (p.G426C) were found to be aberrantly cross-linked by disulfide bonds, giving rise to a 200 kDa form lacking ALP activity. Taken together, our results reveal that the amino acid substitutions at position 426 of TNSALP differentially affect the structure and function of TNSALP, leading to understanding of the molecular and cellular basis of HPP.

Neuromuscular features of hypophosphatasia.

The pathophysiology of the neuromuscular manifestations of hypophosphatasia (HPP) remains unknown. Pyridoxine-sensitive seizures characterize severe forms of infantile HPP. Young children and infants affected with severe forms of HPP, but also adults often present with myopathy characterized by hypotonia or muscle weakness. Chronic pain, of unclear mechanism is also often present. Tissue-non-specific alkaline phosphatase (Alkaline Phosphatase-Liver/Bone/Kidney [ALPL]) is expressed in brain neuronal cell and in muscle cells during development and adulthood. The knockout of the ALPL impacts neuronal functions in animal models. This may occur through metabolic anomalies involving gamma-aminobutyric acid (GABA) and other neurotransmitters via the metabolism of pyridoxal phosphate (vitamin B6) and phosphoethanolamine. In this context, a greater understanding of the neuromuscular pathophysiology of HPP is critical to assess the potential impact of new therapies.

First Korean Case of Infantile Hypophosphatasia with Novel Mutation in ALPL and Literature Review.

Hypophosphatasia is a rare hereditary disorder characterized by defective bone and tooth mineralization and deficiency of tissue non-specific alkaline phosphatase activity. The prognosis for the infantile form is poor, with approximately 50% of patients dying within the first year of life from respiratory failure. We describe the clinical and biochemical findings as well as the molecular analysis of a Korean boy with infantile hypophosphatasia and present a literature review. A 1-month-old boy visited the clinic because of poor feeding, frequent vomiting, hypotonia, and failure to thrive from birth. Laboratory tests revealed high total calcium, low phosphorous, low alkaline phosphatase, low parathyroid hormone, and normal 25-hydroxyvitamin D. Intravenous hydration with normal saline was started, and dietary calcium intake was restricted. Skeletal X-rays showed a markedly increased distance of the anterior fontanelle, impaired mineralization, and rachitic changes in the metaphyses. By Sanger sequencing of the ALPL gene, we identified two heterozygous variants, including a missense (c.334G>A; p.Gly112Ser) and a nonsense (c.1039C>T; p.Gln347*) variant. The c.334G>A (p.Gly112Ser) variant had previously been reported in a patient with lethal type hypophosphatasia, while the nonsense c.1039C>T (p.Gln347*) variant was novel. In the current case, the accurate diagnosis and prompt intervention-including dietary calcium intake restriction, tracheostomy to prevent progression to respiratory failure, and fundoplication with gastrostomy to ensure the administration of adequate calories-seemed to play an important role for avoiding preventable morbidity and premature mortality.

Glycosylation-deficient mutations in tissue-nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNSALP) is a membrane glycoprotein with a proposed role in bone mineralization. Indeed, mutations in TNSALP have been identified in patients with hypophosphatasia (HPP), a genetic disease characterized by hypomineralization of bone and teeth and a deficiency in serum ALP activity. TNSALP has five potential N-glycosylation sites at N140, N230, N271, N303 and N430 by standard nomenclature. A mutation at one of these sites, N430, was recently detected in a patient with infantile HPP. Using site-directed mutagenesis, we demonstrated that TNSALP has five N-glycans in transfected COS-1 cells and that individual single N-glycan deletion mutants of TNSALP retain the dimeric structure required for ALP activity, excluding the possibility that any single N-glycan plays a vital role in the structure and function of TNSALP. However, we found that TNSALP (N430Q) and TNSALP (N430E) mutants, but not a TNSALP (N430D) mutant, failed to form dimers. The TNSALP (N430S) mutant linked to infantile HPP was glycosylation-defective and unable to dimerise, similar to TNSALP (N430Q) and TNSALP (N430E) mutants; therefore, TNSALP (N430S) was established as a severe allele without strong ALP activity. By contrast to individual single N-glycan deletion mutants, TNSALP devoid of all five N-glycans was present to a much lesser extent than wild-type TNSALP in transfected cells, possibly reflecting its instability. A comprehensive analysis of a series of multiple N-glycan depletion mutants in TNSALP revealed that three N-glycans on N230, N271 and N303 were the minimal requirement for the structure and function of TNSALP and a prerequisite for its stable expression in a cell.

Alkaline Phosphatase and Hypophosphatasia.

Hypophosphatasia (HPP) results from ALPL mutations leading to deficient activity of the tissue-non-specific alkaline phosphatase isozyme (TNAP) and thereby extracellular accumulation of inorganic pyrophosphate (PPi), a natural substrate of TNAP and potent inhibitor of mineralization. Thus, HPP features rickets or osteomalacia and hypomineralization of teeth. Enzyme replacement using mineral-targeted TNAP from birth prevented severe HPP in TNAP-knockout mice and was then shown to rescue and substantially treat infants and young children with life-threatening HPP. Clinical trials are revealing aspects of HPP pathophysiology not yet fully understood, such as craniosynostosis and muscle weakness when HPP is severe. New treatment approaches are under development to improve patient care.

Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia.

Hypophosphatasia (HPP) is a genetic disease characterized by defective calcification of hard tissues such as bone and teeth accompanying deficiency of serum alkaline phosphatase (ALP) activity. Its development results from various mutations in the ALPL gene encoding tissue-nonspecific ALP (TNSALP). HPP is known to be transmitted in an autosomal recessive or autosomal dominant manner. A point mutation (c.323C>T) in the ALPL gene leading to a proline to leucine substitution at position 108 of TNSALP was first reported in a patient diagnosed with odonto-HPP (M Herasse et al., J Med Genet 2003;40:605-609), although the effects of this mutation on the TNSALP molecule have not been elucidated. To understand the molecular basis of this dominantly transmitted HPP, we first characterized TNSALP (P108L) by expressing it in COS-1 cells transiently. In contrast to wild-type TNSALP (WT), TNSALP (P108L) showed virtually no ALP activity. When coexpressed with TNSALP (WT), TNSALP (P108L) significantly inhibited the enzyme activity of TNSALP (WT), confirming that this mutant TNSALP exerts a dominant negative effect on TNSALP (WT). Using immunofluorescence and digestion with phosphatidylinositol-specific phospholipase C, we demonstrated that TNSALP (P108L) was anchored to the cell surface via glycosylphosphatidylinositol-like TNSALP (WT) in a Tet-On CHO cell expression system. Consistent with this, TNSALP (P108L) acquired endo-ß-N-acetylglucosaminidase H resistance and sialic acids, as evidenced by glycosidase treatments. Importantly, TNSALP (WT) largely formed a functional dimeric structure, while TNSALP (P108L) was found to be present as a monomer in the cell. This indicates that the molecular structure of TNSALP is affected by a missense mutation at position 108, which is in contact with the active site, such that it no longer assembles into the functional dimeric form. Collectively, these results may explain why TNSALP (P108L) loses its ALP activity, even though it is able to gain access to the cell surface.