Complex intrinsic abnormalities in osteoblast lineage cells of X-linked hypophosphatemia: Analysis of human iPS cell models generated by CRISPR/Cas9-mediated gene ablation.

X-linked hypophosphatemia (XLH) is caused by inactivating variants of the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Although the overproduction of fibroblast growth factor 23 (FGF23) is responsible for hypophosphatemia and impaired vitamin D metabolism, the pathogenesis of XLH remains unclear. We herein generated PHEX-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene ablation to an iPS clone derived from a healthy male, and analyzed PHEX-KO iPS cells with deletions extending from exons 1 to 3 and frameshifts by inducing them to differentiate into the osteoblast lineage. We confirmed the increased production of FGF23 in osteoblast lineage cells differentiated from PHEX-KO iPS cells. In vitro mineralization was enhanced in osteoblast lineage cells from PHEX-KO iPS cells than in those from isogenic control iPS cells, which reminded us of high bone mineral density and enthesopathy in patients with XLH. The extracellular level of pyrophosphate (PPi), an inhibitor of mineralization, was elevated, and this increase appeared to be partly due to the reduced activity of tissue non-specific alkaline phosphatase (TNSALP). Osteoblast lineage cells derived from PHEX-KO iPS cells also showed the increased expression of multiple molecules such as dentine matrix protein 1, osteopontin, RUNX2, FGF receptor 1 and early growth response 1. This gene dysregulation was similar to that in the osteoblasts/osteocytes of Phex-deficient Hyp mice, suggesting that common pathogenic mechanisms are shared between human XLH and Hyp mice. Moreover, we found that the phosphorylation of CREB was markedly enhanced in osteoblast lineage cells derived from PHEX-KO iPS cells, which appeared to be associated with the up-regulation of the parathyroid hormone related protein gene. PHEX deficiency also affected the response of the ALPL gene encoding TNSALP to extracellular Pi. Collectively, these results indicate that complex intrinsic abnormalities in osteoblasts/osteocytes underlie the pathogenesis of human XLH.

The Effect of Asfotase Alfa on Plasma and Urine Pyrophosphate Levels and Pseudofractures in a Patient With Adult-Onset Hypophosphatasia.

Hypophosphatasia (HPP) is an inherited disease caused by variants of the ALPL gene encoding tissue-nonspecific alkaline phosphatase. Adult-onset HPP (adult HPP), known as a mild form of HPP, develops symptoms involving osteomalacia after the age of 18 years. Asfotase alfa (AA) is a modulated recombinant human alkaline phosphatase (ALP) that has been established as a first-line therapy for severe forms of HPP, such as perinatal and infantile forms. We described a 64-year-old female who presented with pseudofractures in bilateral femur diaphyses and impaired mobility. Low serum ALP activity and a high concentration of urine phosphoethanolamine indicated the diagnosis of HPP, which was confirmed by the identification of a homozygous variant in the ALPL gene (c.319G > A; p.Val107Ile). An in vitro transfection experiment to measure the ALP activity of this novel variant protein was performed, resulting in 40% of the residual enzymatic activity compared with the wild type. AA was initiated to facilitate the union of pseudofracture and to improve mobility. After 6 months, radiographic images revealed the disappearance of fracture lines, and improvement of ambulatory ability was confirmed by the 6-minute walk test (525 to 606 m). The EQ-5D-5L index was also improved (0.757 to 0.895). Within a follow-up period, the levels of urine pyrophosphate corrected by urine creatinine (uPPi/Cre) declined in parallel with the level of plasma PPi (plasma PPi: 6.34 to 1.04 µM, uPPi/Cre: 226.8 to 75.4 nmol/mg). The beneficial effect of AA on pseudofracture healing in adult HPP was presented, although the application of AA should be restricted to patients exhibiting relatively severe manifestations. In addition, a novel pathogenic variant of the ALPL gene was identified with the supportive result of functional analysis. Furthermore, when monitoring patients with HPP treated with AA, uPPi/Cre might be a convenient substitute for plasma PPi, which requires immediate filtration after blood sampling. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

A unique case of childhood hypophosphatasia caused by a novel heterozygous 51-bp in-frame deletion in the ALPL 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.

Growth-related skeletal changes and alterations in phosphate metabolism.

Serum inorganic phosphate (Pi) levels are higher in children than in adults; however, the underlying mechanisms remain unclear. Therefore, we herein attempted to elucidate the mechanisms altering Pi metabolism from youth to adulthood using 4-week-old (young) and 12-week-old (adult) mice. Despite higher serum Pi levels, serum fibroblast growth factor 23 (FGF23) levels were lower in young mice, and the amount of FGF23 in bone tended to increase from youth to adulthood. Increases in serum FGF23 levels during growth were associated with the up- and down-regulation of the renal expression of Cyp24a1 encoding vitamin D-24-hydroxylase and Slc34a3 encoding the type IIc sodium/phosphate (Na+/Pi) co-transporter, respectively, suggesting an enhancement in the FGF23-mediated bone-kidney axis from youth to adulthood. We then isolated osteoblasts and osteocytes from young and adult mice and compared the expression of genes involved in Pi metabolism and/or mineralization. In contrast to the growth-related increase in Fgf23 expression, the expression of some genes, including the dentin matrix protein 1 (Dmp1) and phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) markedly decreased from youth to adulthood. The down-regulation of Dmp1 and Phex may contribute to growth-related increases in FGF23. The responses of isolated osteoblasts and osteocytes to high Pi levels also markedly differed between young and adult mice. Treatment of isolated osteocytes with high Pi increased the production of FGF23 in adult mice but not in young mice. These results indicate a close relationship between skeletal changes from youth to adulthood and an alteration in Pi metabolism, and provide insights into the mechanisms by which osteoblasts and osteocytes maintain Pi homeostasis.

Clonal osteoblastic cell lines with CRISPR/Cas9-mediated ablation of Pit1 or Pit2 show enhanced mineralization despite reduced osteogenic gene expression.

Multiple actions of extracellular Pi on the skeletal cells are likely to be partly mediated by type III sodium/phosphate (Na+/Pi) cotransporters Pit1 and Pit2, although the details are not fully understood. In the current study, to determine the roles of Pit1 and Pit2 in osteoblasts, we generated Pit1-knockout (KO) and Pit2-KO osteoblastic cells by applying CRISPR/Cas9 genome editing to an osteoblastic cell line MC3T3-E1 subclone 4. The extracellular Pi level was increased in the Pit1-KO and Pit2-KO clones due to the reduced Pi uptake. Interestingly, in vitro mineralization was accelerated in the Pit1-KO and Pit2-KO clones, although the induction of the expression of osteogenic marker genes was suppressed. In the cells before mineralization, extracellular levels of pyrophosphate (PPi) and adenosine triphosphate (ATP) were increased in the Pit1-KO and Pit2-KO clones, which might be attributable to the reduced expression and activity of tissue-nonspecific alkaline phosphatase (TNSALP). A 24-h treatment with high Pi reduced the expression and activity of TNSALP, suggesting that the suppression of TNSALP in the Pit1-KO and Pit2-KO clones was caused by the increased availability of extracellular Pi. Lentiviral gene transfer of Pit1 and Pit2 restored the changes observed in Pit1-KO and Pit2-KO clones, respectively. The expressions of P2Y2 and P2X7 which encode receptors for extracellular ATP were altered in the Pit1-KO and Pit2-KO clones, suggesting an influence on purinergic signaling. In mineralized cells after long-term culture, intracellular levels of PPi and ATP were higher in the Pit1-KO and Pit2-KO clones. Taken together, ablation of Pit1 or Pit2 in this osteoblastic cell model led to accelerated mineralization, suppressed TNSALP and altered the levels of extracellular and intracellular PPi and ATP, which might be partly mediated by changes in the availability of extracellular Pi.

Novel mutation in the ALPL gene with a dominant negative effect in a Japanese family.

INTRODUCTION: Hypophosphatasia (HPP) is caused by mutations in the ALPL gene encoding tissue nonspecific alkaline phosphatase (TNSALP) and inherited in either an autosomal recessive or autosomal dominant manner. It is characterized clinically by defective mineralization of bone, dental problems, and low serum ALP levels. In the current report, we demonstrate a novel mutation in the ALPL gene (c.244G > A p.Gly82Arg) in a Japanese family with low serum ALP levels. MATERIALS AND METHODS: The ALPL gene analysis using hybridization capture-based next-generation sequencing was performed. The expression plasmids of the wild type and mutated TNSALP were introduced into COS-7 cells. The enzymatic activity of ALP in the cell lysates was measured using p-nitrophenylphosphate as a substrate. RESULTS: TNSALP with the novel ALPL mutation (c.244G > A p.Gly82Arg) completely lost its enzymatic activity and suppressed that of wild-type TNSALP, corroborating its dominant negative effect. The diagnosis of autosomal dominant HPP was confirmed in three members of the family. CONCLUSION: Our approach would help to avoid the inappropriate use of bone resorption inhibitors for currently mis- or under-diagnosed HPP, given that the presence of further, yet undetected mutations of the ALPL gene are plausible.

Hypophosphatasia in Japan: ALPL Mutation Analysis in 98 Unrelated Patients.

Hypophosphatasia (HPP) is highly variable in clinical expression and is generally classified into six subtypes. Although it would be beneficial to be able to predict the clinical course from the ALPL genotype, studies on this issue are limited. Here, we aimed to clarify the features of Japanese HPP and the relationships between genotype and clinical manifestations. We analyzed 98 unrelated Japanese patients to investigate the percentage of each clinical form, frequently detected mutations, and the relationship between the genotype and phenotype. Some of the identified mutants were characterized by transfection experiments. Perinatal severe form was the most frequent (45.9%), followed by perinatal benign form (22.4%). Among the 196 alleles, p.Leu520ArgfsX86 (c.1559delT) was detected in 89 alleles, and p.Phe327Leu (c.979T>C) was identified in 23 alleles. All of the homozygotes for p.Leu520ArgfsX86 were classified into perinatal severe form, and patients carrying p.Phe327Leu in one of the alleles were classified into perinatal benign or odonto HPP. Twenty of the 22 patients with perinatal benign HPP were compound heterozygous for p.Phe327Leu and another mutation. Most patients with odonto HPP were found to be monoallelic heterozygotes for dominant-negative mutations or compound heterozygotes with mutants having residual activity. The high prevalence of p.Leu520ArgfsX86 and p.Phe327Leu mutations might underlie the high rate of perinatal severe and perinatal benign forms, respectively, in Japanese HPP. Although ALPL genotyping would be beneficial for predicting the clinical course to an extent, the observed phenotypical variability among patients sharing the same genotypes suggests the presence of modifiers.

Findings of amplitude-integrated electroencephalogram recordings and serum vitamin B6 metabolites in perinatal lethal hypophosphatasia during enzyme replacement therapy.

Hypophosphatasia (HPP) is a rare disorder caused by low serum tissue non-specific alkaline phosphatase (ALP) activity due to hypomorphic mutations in the ALPL gene. HPP is characterized by defective bone mineralization. It frequently accompanies pyridoxine-responsive seizures. Because alkaline phosphatase change pyridoxal 5' phosphate (PLP) into pyridoxal (PL), which can cross the blood brain barrier and regulates inhibitory neurotransmitter gamma-aminobutyric acid. The female patient was born at a gestational age of 37 weeks 2 days. She presented severe respiratory disorder due to extreme thoracic hypoplasia. With the extremely low serum ALP value (14 IU/L), she was clinically diagnosed as HPP. The diagnosis was confirmed with genetic testing. On day1, the subclinical seizures were detected by aEEG. Together with enzyme replacement therapy by asfotase alfa, pyridoxine hydrochloride was administered, then the seizures were rapidly controlled. While confirming that there was no seizure by aEEG monitoring, pyridoxine hydrochloride was gradually discontinued after 1 month. Before administration of pyridoxine hydrochloride, PL was extremely low (4.7 nM) and PLP was increased (1083 nM). After the withdrawal, PL was increased to 84.9 nM only by enzyme replacement. Monitoring with aEEG enabled early intervention for pyridoxine responsive seizures. Confirming increased serum PL concentration is a prudent step in determining when to reduce or discontinue pyridoxine hydrochloride during enzyme replacement therapy.

CREB activation in hypertrophic chondrocytes is involved in the skeletal overgrowth in epiphyseal chondrodysplasia Miura type caused by activating mutations of natriuretic peptide receptor B.

Natriuretic peptide receptor B (NPRB) produces cyclic guanosine monophosphate (cGMP) when bound by C-type natriuretic peptide (CNP). Activating mutations in NPRB cause a skeletal overgrowth disorder, which has been named epiphyseal chondrodysplasia, Miura type (ECDM; OMIM #615923). Here we explored the cellular and molecular mechanisms for the skeletal overgrowth in ECDM using a mouse model in which an activating mutant NPRB is specifically expressed in chondrocytes. The mutant mice (NPRB[p.V883M]-Tg) exhibited postnatal skeletal overgrowth and increased cGMP in cartilage. Both endogenous and transgene-derived NPRB proteins were localized at the plasma membrane of hypertrophic chondrocytes. The hypertrophic zone of growth plate was thickened in NPRB[p.V883M]-Tg. An in vivo BrdU-labeling assay suggested that some of the hypertrophic chondrocytes in NPRB[p.V883M]-Tg mice continued to proliferate, although wild-type (WT) chondrocytes stopped proliferating after they became hypertrophic. In vitro cell studies revealed that NPRB activation increased the phosphorylation of cyclic AMP-responsive element binding protein (CREB) and expression of cyclin D1 in matured chondrocytes. Treatment with cell-permeable cGMP also enhanced the CREB phosphorylation. Inhibition of cyclic adenosine monophosphate (cAMP)/protein kinase A pathway had no effects on the CREB phosphorylation induced by NPRB activation. In immunostaining of the growth plates for the proliferation marker Ki67, phosphorylated CREB and cyclin D1, most signals were similarly observed in the proliferating zone in both genotypes, but some cells in the hypertrophic zone of NPRB[p.V883M]-Tg were also positively stained. These results suggest that NPRB activation evokes its signal in hypertrophic chondrocytes to induce CREB phosphorylation and make them continue to proliferate, leading to the skeletal overgrowth in ECDM.

Discordant fetal phenotype of hypophosphatasia in two siblings.

Hypophosphatasia (HPP) is an autosomal recessive metabolic disorder with impaired bone mineralization due to mutations in the ALPL gene. The genotype-phenotype correlation of this disorder has been widely described. Here, we present two affected siblings, whose fetal phenotypes were discordant. A 31-year-old Japanese woman, G0P0, was referred to our institution because of fetal micromelia. After obstetric counseling, the pregnancy was terminated at 21 weeks' gestation. Post-mortem radiographs demonstrated severely defective mineralization of the skeleton. The calvarial, spinal, and tubular bones were mostly missing. Only the occipital bones, mandible, clavicles, ribs, one thoracic vertebra, ilia, and tibia were relatively well ossified. The radiological findings suggested lethal HPP. Genetic testing for genomic DNA extracted from the umbilical cord identified compound heterozygous mutations in the ALPL gene (c.532T>C, p.Y178H; c.1559delT, p.Leu520Argfs*86). c.532T>C was a novel variant showing no residual activity of the protein by the functional analysis. The parents were heterozygous carriers. In the next pregnancy, biometric values on fetal ultrasonography at 20 and 26 weeks' gestation were normal. At 34 weeks, however, a small chest and shortening of distal long bones came to attention. The neonate delivered at 41 weeks showed serum ALP of <5U/L. Radiological examination showed only mild thoracic hypoplasia and metaphyseal mineralization defects of the long bones. ALP replacement therapy was introduced shortly after birth, and the neonate was discharged at day 22 without respiratory distress. Awareness of discordant fetal phenotypes in siblings with HPP precludes a diagnostic error, and enables early medical intervention to mildly affected neonates.

Cover Image, Volume 176A, Number 1, January 2018.

The cover image, by Satoru Ikenoue et al., is based on the Clinical Report Discordant fetal phenotype of hypophosphatasia in two siblings, DOI: 10.1002/ajmg.a.38531.

Extracellular Phosphate Induces the Expression of Dentin Matrix Protein 1 Through the FGF Receptor in Osteoblasts.

Dentin matrix protein 1 (Dmp1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1, fibroblast growth factor 2 (Fgf2), and Fgf receptor 1 (Fgfr1). A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 (Egr1) before the up-regulation of Dmp1. These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. J. Cell. Biochem. 118: 1151-1163, 2017. © 2016 Wiley Periodicals, Inc.

Reversible Deterioration in Hypophosphatasia Caused by Renal Failure With Bisphosphonate Treatment.

Hypophosphatasia is an inborn error of metabolism caused by mutations in the ALPL gene. It is characterized by low serum alkaline phosphatase (ALP) activity and defective mineralization of bone, but the phenotype varies greatly in severity depending on the degree of residual enzyme activity. We describe a man with compound heterozygous mutations in ALPL, but no previous bone disease, who suffered numerous disabling fractures after he developed progressive renal failure (for which he eventually needed dialysis treatment) and was prescribed alendronate treatment. A bone biopsy showed marked osteomalacia with low osteoblast numbers and greatly elevated pyrophosphate concentrations at mineralizing surfaces. In vitro testing showed that one mutation, T117H, produced an ALP protein with almost no enzyme activity; the second, G438S, produced a protein with normal activity, but its activity was inhibited by raising the media phosphate concentration, suggesting that phosphate retention (attributable to uremia) could have contributed to the phenotypic change, although a pathogenic effect of bisphosphonate treatment is also likely. Alendronate treatment was discontinued and, while a suitable kidney donor was sought, the patient was treated for 6 months with teriparatide, which significantly reduced the osteomalacia. Eighteen months after successful renal transplantation, the patient was free of symptoms and the scintigraphic bone lesions had resolved. A third bone biopsy showed marked hyperosteoidosis but with plentiful new bone formation and a normal bone formation rate. This case illustrates how pharmacological (bisphosphonate treatment) and physiologic (renal failure) changes in the "environment" can dramatically affect the phenotype of a genetic disorder.

Interleukin-1-induced acute bone resorption facilitates the secretion of fibroblast growth factor 23 into the circulation.

Fibroblast growth factor 23 (FGF23), a central regulator of phosphate and vitamin D metabolism, is mainly produced by osteocytes in bone and exerts its effects on distant organs. Despite its endocrine function, the mechanism controlling serum FGF23 levels is not fully understood. Here we tested the hypothesis that osteoclastic bone resorption may play a role in regulating circulating levels of FGF23, using a mouse model where injections of interleukin (IL)-1ß into the subcutaneous tissue over the calvaria induced rapid bone resorption. A significant amount of FGF23 was detected in the extracts from mouse bones, which supports the idea that FGF23 stays in bone for a while after its production. IL-1ß-induced bone resorption was associated with elevated serum FGF23 levels, an effect abolished by pre-treatment with pamidronate. Fgf23 expression was not increased in either the calvariae or tibiae of IL-1ß-injected mice, which suggests that IL-1ß facilitated the entry of FGF23 protein into circulation by accelerating bone resorption rather than increasing its gene expression. The direct effect of IL-1ß on bone was confirmed when it increased FGF23 levels in the conditioned media of mouse calvariae in organ culture. Repeated treatment of the cultured calvariae with IL-1ß led to a refractory phase, where FGF23 was not mobilized by IL-1ß anymore. Consistent with the in vivo results, treatment with IL-1ß failed to increase Fgf23 mRNA in isolated primary osteocytes and osteoblasts. These results suggest that FGF23 produced by osteocytes remains in bone, and that rapid bone resorption facilitates its entry into the bloodstream.

Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

Elevated fibroblast growth factor 23 exerts its effects on placenta and regulates vitamin D metabolism in pregnancy of Hyp mice.

Fibroblast growth factor 23 (FGF23) functions in an endocrine fashion and requires α-Klotho to exert its effects on the target organs. We have recently demonstrated that the human placenta also expresses α-Klotho, which led us to hypothesize that FGF23 may exert effects on the placenta. Immunohistochemical analysis demonstrated the expression of FGF receptor 1 (FGFR1) as well as that of α-Klotho in the feto-maternal interface of both mouse and human normal-term placentas, which suggested that these areas might be receptive to FGF23. Therefore, we next investigated whether FGF23 has some roles in the placenta using Hyp mice with high levels of circulating FGF23. Hyp and wild-type (WT) females were mated with WT males, and the mothers and their male fetuses were analyzed. FGF23 levels in Hyp mothers were elevated. FGF23 levels were about 20-fold higher in Hyp fetuses than in Hyp mothers, whereas WT fetuses from Hyp mothers exhibited low levels of FGF23, as did fetuses from WT mothers. We analyzed the placental gene expression and found that the expression of Cyp24a1 encoding 25OHD-24-hydroxylase, a target gene for FGF23 in the kidney, was increased in the placentas of fetuses from Hyp mothers compared with fetuses from WT mothers. In an organ culture of WT placentas, treatment with plasma from Hyp mothers markedly increased the expression of Cyp24a1, which was abolished by the simultaneous addition of anti-FGF23 neutralizing antibody. The direct injection of recombinant FGF23 into WT placentas induced the expression of Cyp24a1. The increase in the placental expression of Cyp24a1 in fetuses from Hyp mothers resulted in decreased plasma 25-hydroxyvitamin D levels. These results suggest that increased levels of circulating FGF23 in pathological conditions such as Hyp mice exerts direct effects on the placenta and affects fetal vitamin D metabolism via the regulation of Cyp24a1 expression.

Benign prenatal hypophosphatasia: a treatable disease not to be missed.

Prenatal bowing of the long bones is often associated with severe bone dysplasias. We report a child who presented marked bowing of the long bones at birth but showed a relatively benign postnatal course with spontaneous improvement of bowing. The fetal imaging showed normal skeletal mineralization and normal chest and abdominal circumferences despite the limb bowing and shortening. Decreased serum alkaline phosphatase activity and elevated urine phosphoethanolamine was biochemically evident, and compound heterozygous mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene were identified, which confirmed the diagnosis of a benign form of prenatal hypophosphatasia. Benign prenatal hypophosphatasia should be considered in the differential diagnosis of congenital bowing of the long bones.

Sodium-coupled neutral amino acid transporter 4 functions as a regulator of protein synthesis during liver development.

AIM: The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes. METHODS: HNF4α was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4α was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression. RESULTS: The expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4α. Transactivation of AP2 was upregulated by HNF4α in HeLa cells without endogenous HNF4α. EMSA has demonstrated that HNF4α directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts. CONCLUSION: SNAT4 functions downstream of HNF4α and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.

A case of autosomal dominant osteopetrosis type II with a novel TCIRG1 gene mutation.

Osteopetrosis is a rare genetic disorder characterized by increased bone mineral density (BMD) due to osteoclast failure. T-cell immune regulator 1 (TCIRG1) plays crucial roles on osteoclast function, and its mutation causes autosomal recessive osteopetorosis. However, mutations in TCIRG1 have never been identified in autosomal dominant osteopetrosis (ADO). A 3-year-old boy was first presented to the clinic because of spontaneous radius and femur fractures. He has optic atrophy. The areal BMD at the lumbar spine was 1274 g/cm2 (233% of normal). Laboratory tests revealed no remarkable abnormal findings, including anemia, except for extremely elevated serum tartrate-resistant acid phosphatase-5b (14,600 mU/dL). Radiographically, the skull base, pelvis, and vertebrae showed a focal sclerosis. Genetic analysis revealed a novel de novo heterozygous missense mutation (His242Arg). Taken together with the mutation, his mild clinical features were diagnosed as ADO. This case implies that TCIRG1 could become a genetic candidate for ADO in addition to malignant forms such as ARO.