"Atypical femoral fractures" during bisphosphonate exposure in adult hypophosphatasia

We report a 55-year-old woman who suffered atypical subtrochanteric femoral fractures (ASFFs) after 4 years of exposure to alendronate and then zolendronate given for "osteoporosis." Before alendronate treatment, she had low bone mineral density. After several months of therapy, metatarsal stress fractures began. Bisphosphonate (BP) administration was stopped following the ASFFs, and the adult form of hypophosphatasia (HPP) was diagnosed from low serum alkaline phosphatase (ALP) activity, high endogenous levels of two natural substrates for the "tissue-nonspecific" isoenzyme of ALP (TNSALP), and a heterozygous mutation within the gene that encodes this enzyme. Experience with other HPP families showed that her mutation (Arg71His) with a second defective TNSALP allele can cause severe HPP in infancy, and when heterozygous can cause mild HPP featuring premature loss of deciduous teeth in children. Because the skeletal disease of HPP results from extracellular accumulation of the TNSALP substrate inorganic pyrophosphate (PPi) and its inhibitory effect on mineralization, perhaps HPP patients or carriers will have adverse effects from BPs. BPs are analogues of PPi and can suppress bone turnover but also deactivate TNSALP. Our report is the first of BP exposure preceding ASFFs in adult HPP. To explore a potential role for TNSALP deactivation in ASFFs, mutation analysis of TNSALP should be studied in a cohort of these patients. Meanwhile, clinicians must suspect HPP when clinical or laboratory clues include premature loss of primary dentition, pseudofractures or recurrent poorly healing metatarsal stress fractures, a family history suggestive of HPP, or low serum ALP activity. If HPP is documented, BP treatment might be avoided. To establish the diagnosis of HPP, assays for two natural substrates for TNSALP and TNSALP mutation analysis are available in commercial laboratories. With positive findings, radiological or bone biopsy evidence of acquired osteomalacia would indicate the adult form of this inborn-error-of-metabolism.     

Enzyme Replacement Therapy for Murine Hypophosphatasia

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss‐of‐function mutation(s) within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PP_i), an inhibitor of mineralization, and pyridoxal 5′‐phosphate (PLP), a co‐factor form of vitamin B₆. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B₆‐dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one‐step purification and a deca‐aspartate sequence (D₁₀) for targeting to mineralizing tissue (sALP‐FcD₁₀). TNALP‐null mice (Akp2^?/?), an excellent model for infantile HPP, were treated from birth using sALP‐FcD₁₀. Short‐term and long‐term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP‐FcD₁₀ for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP‐FcD₁₀ activity, calcium, PP_i, and pyridoxal, as well as skeletal and dental manifestations using radiography, μCT, and histomorphometry. Results: Akp2^?/? mice receiving high‐dose sALP‐FcD₁₀ grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PP_i, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone‐targeted, recombinant form of human TNALP prevents infantile HPP in Akp2^?/? mice.     

Pyridoxine-responsive seizures as the first symptom of infantile hypophosphatasia caused by two novel missense mutations (c.677T>C, p.M226T; c.1112C>T, p.T371I) of the tissue-nonspecific alkaline phosphatase gene

Pyridoxine-responsive seizures (PRS) and the role of pyridoxine (PN, vitamin B₆) in hypophosphatasia (HPP) are incompletely understood. Typically, PRS and HPP are rare, independent, metabolic disorders. In PRS, seizures resist standard anticonvulsants apart from PN, yet have a good prognosis. In HPP, inactivation of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP) impairs skeletal mineralization and causes rickets in infants that can be fatal. Here, we report a 7-month-old girl, newly diagnosed with infantile HPP, who presented as a neonate with PRS but without bony abnormalities. Analysis of biogenic amines in cerebrospinal fluid (CSF) suggested brain pyridoxal 5′-phosphate (PLP) deficiency, although PLP in CSF was not decreased. She had normal cognitive milestones but failure to thrive and rickets. Nearly undetectable serum ALP activity, elevated plasma PLP and urinary phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi) levels, hypercalcemia, hypercalciuria and nephrocalcinosis were consistent with infantile HPP. Only prednisolone reduced serum calcium levels. Despite improved growth and weight gain, she developed rib fractures and died from respiratory failure at age 9 months. Sequence analysis of the TNSALP gene revealed novel missense mutations in exon 7 (c.677T > C, p.M226T) and exon 10 (c.1112C > T, p.T371I). Our patient demonstrated that PRS in neonates may not necessarily be “idiopathic”; instead, such seizures can be caused by severe HPP that becomes clinically apparent later in infancy. The pathophysiology of PRS in HPP differs from the three other genetic defects known to cause PRS, but all may lead to brain PLP deficiency reducing seizure thresholds. All reported HPP patients with neonatal seizures died within 18 months of birth, suggesting that PRS is an indicator of HPP severity and lethal prognosis. We recommend that assessment of any neonate with PRS should include measurement of serum ALP activity.     

Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (-/-) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of approximately 50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at approximately 10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.     

Calcific Periarthritis as the Only Clinical Manifestation of Hypophosphatasia in Middle‐Aged Sisters

Hypophosphatasia (HPP) is the inborn error of metabolism that features low serum alkaline phosphatase (ALP) activity caused by loss‐of‐function mutation(s) within the gene for the tissue nonspecific isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP substrate and inhibitor of mineralization, leads frequently to premature tooth loss and often to rickets or osteomalacia. In affected adults, the excess PPi sometimes also causes calcium pyrophosphate dihydrate (CPPD) deposition, PPi arthropathy, or pseudogout, or seemingly paradoxical deposition of hydroxyapatite crystals in ligaments or around joints when the condition is called calcific periarthritis (CP). We report three middle‐aged sisters with CP as the only clinical manifestation of HPP. Each presented during early adult life with recurrent episodes of pain principally around the shoulders, elbows, wrists, hips, or Achilles tendon. Otherwise, they were in good health, including no history of unusual dental disease, fractures, or pseudofractures. Calcific deposits were identified in symptomatic areas principally by ultrasonographic assessment but also confirmed radiographically. All three sisters had low serum levels of total and bone‐specific ALP, hyperphosphatemia, and increased serum concentrations of the TNSALP substrate pyridoxal 5′‐phosphate together characteristic of HPP. Mutation analysis revealed that each carried a single unique 18‐bp duplication within TNSALP (c.188_205dup18, p.Gly63_Thr68dup) as did two of their healthy sons and their mother, who was without signs of CPPD deposition or CP but had knee osteoarthritis. We find that CP can be the only complication of HPP in adults. Thus, multiple juxta‐articular deposits of hydroxyapatite causing CP may be a useful sign of HPP, especially when the CP is familial. © 2014 American Society for Bone and Mineral Research.     

Dose response of bone-targeted enzyme replacement for murine hypophosphatasia

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2mg/kg for 43days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED(80) (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PP(i) concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP in patients, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP to mice. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials.     

Prolonged survival and phenotypic correction of Akp2−/− hypophosphatasia mice by lentiviral gene therapy

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue‐nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2^?/?) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP‐deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone‐targeted form of TNALP was injected into the jugular vein of newborn Akp2^?/? mice. We found that alkaline phosphatase activity in the plasma of treated Akp2^?/? mice increased and remained at high levels throughout the life of the animals. The treated Akp2^?/? mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2^?/? mice, and X‐ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period. © 2011 American Society for Bone and Mineral Research.     

Hypophosphatasia: Validation and expansion of the clinical nosology for children from 25 years experience with 173 pediatric patients

Hypophosphatasia (HPP) is caused by loss-of-function mutation(s) within the gene TNSALP that encodes the “tissue-nonspecific” isoenzyme of alkaline phosphatase (TNSALP). In HPP, inorganic pyrophosphate, an inhibitor of mineralization and substrate for TNSALP, accumulates extracellularly often leading to rickets or osteomalacia and tooth loss, and sometimes to craniosynostosis and calcium crystal arthropathies. HPP's remarkably broad-ranging expressivity spans stillbirth from profound skeletal hypomineralization to adult-onset dental problems or arthropathies without bone disease, which is largely explained by autosomal recessive versus autosomal dominant transmission from among several hundred, usually missense, TNSALP mutations. For clinical purposes, this expressivity has been codified according to absence or presence of skeletal disease and then patient age at presentation and diagnosis. Pediatric patients are reported principally with “odonto”, “childhood”, “infantile”, or “perinatal” HPP. However, this nosology has not been tested using a cohort of patients, and the ranges of the clinical and laboratory findings have not been defined and contrasted among these patient groups.To evaluate the extant nosology for HPP in children, we assessed our 25 years experience with 173 pediatric HPP patients. Data were exclusively from inpatient studies. The childhood form of HPP was further designated “mild” or “severe”. Here, we focused on demographic, clinical, and dual-energy X-ray absorptiometry parameters compared to data from healthy American children.The 173-patient cohort comprised 64 individuals with odonto HPP, 38 with mild childhood HPP, 58 with severe childhood HPP, and 13 with infantile HPP. None was a survivor of perinatal HPP. TNSALP analysis revealed a mutation(s) in all 105 probands tested. Thirteen mutations were unique. Most patients represented autosomal dominant inheritance of HPP. Mutant allele dosage generally indicated the disorder's severity. Gender discordance was found for severe childhood HPP; 42 boys versus 16 girls (p = 0.006), perhaps reflecting parental concern about stature and strength. Key disease parameters (e.g., height, weight, numbers of teeth lost prematurely, grip strength, spine and hip bone mineral density) were increasingly compromised as HPP was designated more severe. Although data overlapped successively between the four patient groups, body size (height and weight) differed significantly.Thus, our expanded nosology for HPP in children organizes the disorder's broad-ranging expressivity and should improve understanding of HPP presentation, natural history, complications, and prognosis.     

Hypophosphatasia: Enzyme Replacement Therapy Brings New Opportunities and New Challenges

Hypophosphatasia (HPP) is caused by loss‐of‐function mutation(s) of the gene that encodes the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP). Autosomal inheritance (dominant or recessive) from among more than 300 predominantly missense defects of TNSALP (ALPL) explains HPP's broad‐ranging severity, the greatest of all skeletal diseases. In health, TNSALP is linked to cell surfaces and richly expressed in the skeleton and developing teeth. In HPP,TNSALP substrates accumulate extracellularly, including inorganic pyrophosphate (PPi), an inhibitor of mineralization. The PPi excess can cause tooth loss, rickets or osteomalacia, calcific arthropathies, and perhaps muscle weakness. Severely affected infants may seize from insufficient hydrolysis of pyridoxal 5?‐phosphate (PLP), the major extracellular vitamin B₆. Now, significant successes are documented for newborns, infants, and children severely affected by HPP given asfotase alfa, a hydroxyapatite‐targeted recombinant TNSALP. Since fall 2015, this biologic is approved by regulatory agencies multinationally typically for pediatric‐onset HPP. Safe and effective treatment is now possible for this last rickets to have a medical therapy, but a number of challenges involving diagnosis, understanding prognosis, and providing this treatment are reviewed herein. © 2017 American Society for Bone and Mineral Research.     

Chronic Recurrent Multifocal Osteomyelitis Mimicked in Childhood Hypophosphatasia

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by low serum alkaline phosphatase (ALP) activity caused by inactivating mutations within TNSALP, the gene that encodes the “tissue‐nonspecific” isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate, a TNSALP substrate, inhibits hydroxyapatite crystal growth leading to rickets or osteomalacia. Chronic recurrent multifocal osteomyelitis (CRMO) is the pediatric syndrome of periarticular pain and radiographic changes resembling infectious osteomyelitis but without lesional pathogens. Some consider CRMO to be an autoinflammatory disease. An unrelated boy and girl with the childhood form of HPP suffered chronic, multifocal, periarticular pain, and soft tissue swelling. To investigate this unusual complication, we evaluated their cumulative clinical, biochemical, radiological, and histopathological findings and performed mutation analysis of their TNSALP alleles. The earliest radiographic disturbances were typical of childhood HPP. Subsequently, changes consistent with CRMO developed at sites where there was pain, including lucencies, osteosclerosis, and marked expansion of the underlying metaphyses. Bone marrow edema was shown by MRI. Biopsies of affected bone showed nonspecific histopathological findings and no pathogens. The boy was heterozygous (c.1133A>T, p.D378V) and the girl compound heterozygous (c.350A>G, p.Y117C, c.400_401AC>CA, p.T134H) for different TNSALP missense mutations. Nonsteroidal anti‐inflammatory drugs diminished their pain, which improved or resolved at maturity. HPP should be considered when CRMO is a diagnostic possibility. Metaphyseal radiographic changes and marrow edema associated with periarticular bone pain and soft tissue swelling suggestive of osteomyelitis can complicate childhood HPP.     

Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl−/− Mice

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PP_i), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D₁₀ to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl^−/−) mice, a murine model for severe infantile HPP. Alpl^−/− mice received 3 × 10¹¹ vector genomes/body of AAV8-TNAP-D₁₀ within 5 days postnatal (dpn). AAV8-TNAP-D₁₀ elevated serum ALP activity and suppressed plasma PP_i. Treatment extended life span of Alpl^−/− mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl^−/− mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl^−/− mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl^−/− mice versus controls. Treated Alpl^−/− mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Hypophosphatasia: Natural history study of 101 affected children investigated at one research center

Hypophosphatasia (HPP) is the inborn-error-of-metabolism that features deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Resultant extracellular accumulation of inorganic pyrophosphate, a TNSALP substrate and potent inhibitor of mineralization, typically leads to tooth loss and sometimes to rickets or osteomalacia. HPP's remarkably broad-ranging severity is largely explained by autosomal dominant versus autosomal recessive transmission from among several hundred usually missense mutations positioned throughout the gene that encodes TNSALP. In 2015, our cross-sectional investigation of 173 affected children validated and expanded the clinical nosology commonly used for pediatric HPP.Herein, for the 101 patients in that cohort with longitudinal data, we explored the natural history of pediatric HPP by assessing their z-scores for height and then for weight, grip strength, and bone mineral density (BMD) determined by dual energy X-ray absorptiometry (DXA) also after adjusting for patient height. Eighteen patients contributed to “across” puberty evaluation.According to increasing HPP severity, there were 28 odonto HPP, 28 mild childhood HPP, 37 severe childhood HPP, and 8 infantile HPP patients typically studied from early to mid-childhood. The individual values for each parameter were wide-ranging within, and overlapping between, the four successive patient groups. Final mean/median z-scores, like the published initial values, paralleled the nosology. Longitudinal findings were similar for the boys versus girls and across puberty. Mean/median height z-scores remained constant for all four patient groups. In contrast, mean/median weight z-scores increased with aging, including after height-adjustment, resembling the recent trend for American children. However, excessive weight gain was typically not observed and mean/median values became average for height. Mean/median z-scores calculated routinely for chronologic age did not change for grip strength or for lumbar spine or total hip BMD. However, height-correction of the cohort suggested some worsening of grip strength z-scores and indicated improvement in spine BMD z-scores.Overall, in affected children and adolescents, HPP represents a clinically stable but chronic disorder.     

Elevated skeletal osteopontin levels contribute to the hypophosphatasia phenotype in Akp2(-/-) mice.

Increased levels of ePP(i) in mice deficient in TNALP (i.e., Akp2(-/-)) lead to elevated OPN concentrations. We examined the skeletal phenotype of mice lacking both OPN and TNALP and concluded that the increased OPN levels contribute to the hypophosphatasia phenotype characteristic of Akp2(-/-) mice. We also found that extracellular OPN regulates the PP(i) output by osteoblasts. INTRODUCTION: Akp2(-/-) display mineralization deficiencies characterized by rickets/osteomalacia. This defect has been attributed to the increased levels of extracellular inorganic pyrophosphate (ePP(i)), a substrate of tissue-nonspecific alkaline phosphatase (TNALP) and a potent inhibitor of mineral deposition. Because elevated levels of ePP(i) induce Opn gene expression, the Akp2(-/-) mice also display increased levels of osteopontin (OPN), another inhibitor of mineralization. MATERIALS AND METHODS: Akp2(-/-) mice were bred into the Opn(-/-) line. The resulting double knockout mice were analyzed for skeletal abnormalities by histology and muCT. Calvarial osteoblasts were assayed for their ability to mineralize in vitro and were probed for changes in gene expression. RESULTS: Mice lacking both Akp2 and Opn showed partial normalization at the histological level with regard to mineral deposition and BMD. However, high ePP(i) levels remained in Akp2(-/-) mice. We found that Opn(-/-) mice have themselves elevated levels of ePP(i) attributable to an increase in Enpp1 and Ank expression and a concomitant downregulation of Akp2 expression in Opn(-/-) osteoblasts, but that Opn(-/-) mice have more mineralized osteoid than wildtype (WT) controls despite their elevated ePP(i) levels. Addition of exogenous OPN to Opn(-/-) osteoblasts results in downregulation of Enpp1 and Ank gene expression and a reduction of the PP(i) output by these cells. CONCLUSIONS: Deletion of both Akp2 and Opn can partially rescue the hypomineralized phenotype of Akp2(-/-) mice. However, these double knockout mice do not display corrected ePP(i) levels, and we conclude that regulation of hydroxyapatite deposition requires the coordinated actions of both PP(i) and OPN and that the hypophosphatasia phenotype in Akp2(-/-) mice results from the combined inhibitory action of increased levels of both ePP(i) and OPN. Our data also suggest that the ePP(i)-mediated regulation of OPN and the OPN-mediated regulation of ePP(i) are linked counterregulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePP(i).     

Infantile hypophosphatasia: Enzymatic defect explored with alkaline phosphatase-deficient skin fibroblasts in culture

Summary Evidence that infantile hypophosphatasia may result from defective regulation of an intact structural gene for the tissue nonspecific (bone/liver/kidney) isoenzyme of alkaline phosphatase (TNSALP) was explored by studying physicochemical properties of ALP in sonicates of monolayers of cultured dermal fibroblasts from 7 patients (PT) and 5 age- and sex-matched control (CT) subjects. Both groups had low levels of ALP activity when assayed with 4-methylumbelliferyl phosphate substrate. The mean specific activity of ALP in the PT fibroblasts was markedly subnormal (V_max less than 1% of CT), but apparently not from extracellular loss of enzyme, since defined medium had less ALP activity when conditioned by PT compared to CT cells. Although the mean Km for the sonicate ALP was similar for both groups at pH 10.1, pH optimum, thermal stability and response to several inhibitors appeared to be different. Nevertheless, it seemed that some TNSALP-like enzyme was present in the PT group. Exposure of cells in culture to 5-azacytidine and several putative inducers of ALP failed to increase the enzyme activity in either the PT or CT groups. Had the physicochemical properties of the constitutive (or inducible) ALP been the same in the PT and CT cell groups, the findings would have provided evidence for the generality of our previous observations in one patient which indicated that defective regulation of an intact structural gene for TNSALP could account for hypophosphatasia. Our results do not, however, refute this possibility; e.g., especially minute amounts of typical TNSALP may have been present in our PT fibroblasts but were masked by (1) normal or compensatory expression of trace levels of ALP from a separate gene, (2) heterogeneity of the ALPs in the various PT (or CT) cell lines, or (3) the presence of some additional TNSALP that was different from the constitutive ALP of normal fibroblasts. Indeed, review of the findings for individual cell lines suggested that in one patient with relatively high levels of ALP activity, the physicochemical properties of this ALP was most like the enzyme in CT cells. Although further studies—including use of sensitive immunologic and electrophoretic methods—will be necessary to further characterize the minute amounts of residual ALP(s) in infantile hypophosphatasia tissues, presence of some TNSALP-like ALP in PT fibroblasts indicates that the disorder is not commonly due to a complete gene deletion for TNSALP. Our findings do indicate an especially profound and stable deficiency of typical constitutive TNSALP in fibroblasts in this disorder and suggest, therefore, that these cells might be a valuable model to explore the physiologic role of TNSALP.     

Hypophosphatasia: Nonlethal disease despite skeletal presentation in utero (17 new cases and literature review)

Hypophosphatasia (HPP) is caused by deactivating mutation(s) within the gene that encodes the tissue‐nonspecific isoenzyme of alkaline phosphatase (TNSALP). Patients manifest rickets or osteomalacia and dental disease ranging from absence of skeletal mineralization in utero to only loss of adult dentition. Until recently, HPP skeletal disease in utero was thought to always predict a lethal outcome. However, several reports beginning in 1999 emphasized a benign prenatal form of HPP (BP‐HPP) where skeletal disease detected in utero had a mild postnatal course. Here we describe prenatal and postnatal findings of 17 additional BP‐HPP patients among our 178 pediatric HPP patients. Their findings are compared with those of their siblings with HPP, carrier parents, and others with identical TNSALP mutations. New information concerning 7 previously published BP‐HPP patients accompanies a review of the HPP literature. Among our 17 BP‐HPP patients, prenatal ultrasound showed normal chest or abdominal circumferences where recorded. Sometimes, poor skeletal mineralization, fetal crowding, and third‐trimester improvement were observed. Postnatally, extremity bowing further improved (13 patients). BP‐HPP severity postnatally spanned the “infantile” to “odonto” HPP phenotypes, resembling our patients who harbored identical TNSALP mutation(s). Eight had autosomal dominant (AD) and 9 had autosomal recessive (AR) BP‐HPP. Fourteen of our 15 mothers were HPP carriers or affected. Of the 41 cumulative BP‐HPP patients (24 literature cases meriting a BP‐HPP diagnosis since 1996 plus our 17 patients), 63% had AR BP‐HPP. Maternally transmitted HPP involved 11 of the 13 total AD BP‐HPP probands (p = 0.01), supporting a maternal in utero effect on the baby. Fetal crowding, normal fetal mineralization and chest size, and TNSALP heterozygosity seem to identify BP‐HPP. However, bowed fetal long bones with AR HPP, specific TNSALP mutations, or poor skeletal mineralization before the third trimester do not reliably diagnose HPP lethality. © 2011 American Society for Bone and Mineral Research     

Function of mutant (G1144A) tissue-nonspecific ALP gene from hypophosphatasia.

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild-type and G1144A mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h or 72-h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000 g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p-nitrophenylphosphate as a substrate in 100 mM of 2-amino-2-methyl-1,3-propanediol-HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48-h or 72-h transfection exhibited 0.063 +/- 0.012 U/mg and 0.054 +/- 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock-transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease-causing mutation of the childhood-type HOPS.     

Loss-of-Function Mutations in the ALPL Gene Presenting with Adult Onset Osteoporosis and Low Serum Concentrations of Total Alkaline Phosphatase

Hypophosphatasia (HPP) is a rare inherited disorder characterized by rickets and low circulating concentrations of total alkaline phosphatase (ALP) caused by mutations in ALPL. Severe HPP presents in childhood but milder forms can present in adulthood. The prevalence and clinical features of adult HPP are poorly defined. The aim of this study was to evaluate the prevalence and clinical significance of low serum total alkaline phosphatase (ALP) levels in a clinic-based population of adult osteoporotic patients. We searched for patients with low ALP in a cohort of 3285 patients referred to an osteoporosis clinic over a 10-year period and performed mutation screening of ALPL in those with low ALP (≤40 U/L) on two or more occasions. These individuals were matched with four clinic controls with a normal ALP. We also evaluated the prevalence of low ALP and ALPL mutations in 639 individuals from the general population from the same region. We identified 16/3285 (0.49%) clinic patients with low ALP and 14 (87.5%) had potentially pathogenic variants in ALPL. Eight of these individuals were heterozygous for mutations previously described in HPP and 2 were heterozygous for novel mutations (p.Arg301Trp and p.Tyr101X). These mutations were not found in clinic controls or in the general population. Eight patients with low ALP, including 4 with ALPL mutations, were treated with bisphosphonates for an average of 6.5 years. In these individuals, the rate of fractures during treatment was comparable to that in normal ALP clinic controls who were treated with bisphosphonates. We conclude that heterozygous loss-of-function mutations in ALPL are common in osteoporosis patients with low ALP. Further studies are required to determine how best these individuals should be treated. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.     

Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl^−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl^−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl^−/− mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl^−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.     

Hypophosphatasia in Adults: Clinical Spectrum and Its Association With Genetics and Metabolic Substrates

Background: Hypophosphatasia (HPP) is a rare metabolic bone disorder caused by mutations in the alkaline phosphatase (ALPL) gene, and characterized by low circulating alkaline phosphatase (ALP) levels and bone, muscle, dental and systemic manifestations. In this case series we investigate the clinical spectrum, genetic and biochemical profile of adult HPP patients from the University Hospitals Leuven, Belgium. Methodology: Adults with HPP were identified through medical record review. Inclusion criteria were: (1) age ≥ 16 yr; (2) consecutively low ALP levels not explained by secondary causes; (3) one or more of the following supporting criteria: biochemical evidence of elevated enzyme substrates; subtrochanteric fractures, metatarsal fractures or other typical clinical features; family history of HPP; a known or likely pathogenic ALPL mutation. Results: Nineteen patients met our inclusion criteria (n = 2 infantile, n = 6 childhood, n = 10 adult-onset HPP and one asymptomatic carrier). Fractures and dental abnormalities were the most reported symptoms. Fatigue was reported in n = 7/19 patients (37%), three of which had previously been misdiagnosed as having chronic fatigue syndrome and/or fibromyalgia. Empirical pyridoxine therapy in four patients (without seizures) did not provide symptomatic relief. N = 7/19 patients (37%) were inappropriately treated or planned to be treated with antiresorptive treatment. Two patients developed atypical femoral fractures following exposure to bisphosphonates and/or denosumab. Patients detected by screening were less severely affected, while patients with homozygous or compound heterozygous mutations had the most severe symptoms, significantly lower circulating ALP levels (p = 0.013) and significantly higher pyridoxal-5’-phosphate (p = 0.0018) and urinary phosphoethanolamine (p = 0.0001) concentrations. Conclusions: Screening may detect mainly less severely affected individuals, which may nevertheless avoid misdiagnosis and inappropriate antiresorptive drug exposure. Patients with biallelic mutations had more severe symptoms, significantly lower ALP and higher substrate levels. Whether the latter finding has implications for the classification and treatment of HPP should be investigated further in larger cohorts.     

Enzyme replacement for craniofacial skeletal defects and craniosynostosis in murine hypophosphatasia

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl^−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, vertebral and tooth mineralization defects in Alpl^−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl^−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microscopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl^−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl^−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.