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.

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.

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.

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.