Hypophosphatemic rickets, which is often hereditary, is still under- or misdiagnosed in both children and adults, denying these individuals access to optimal management and genetic counseling. There have been recent calls to compile real-world data and share best practice on these rare conditions to guide clinical decision-making. Here we present eight clinical vignettes of patients with hypophosphatemic rickets encountered in our tertiary pediatric endocrinology practice. We describe the clinical features, genetics, and management of four cases of X-linked hypophosphatemia (PHEX mutations), one each of autosomal recessive hypophosphatemic rickets (DMP1 mutation) and autosomal recessive vitamin D-dependent rickets type 1A (CYP27B1 mutation), and two cases of distal renal tubular acidosis with FOXI1 mutation-associated hypophosphatemic rickets. Our cases prompt consideration of the (i) frequent misdiagnosis of hypophosphatemic rickets in clinical practice and the importance of comprehensive genetic testing; (ii) variable expressivity of the causative mutations; and (iii) a lack of responsiveness and/or compliance to conventional therapy and the value of burosumab in modern management, provided access is equitable. These cases highlight common real-world themes and challenges to managing patients presenting with these diverse conditions, especially the burden of disease hidden by misdiagnosis. In sharing these cases, we hope to raise awareness of these conditions, promote best practice in genetic diagnosis and management, and further advocate for reimbursement equity for the best available therapies.

Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands\' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.

Osteocytes have recently been identified as a new regulator of bone remodeling, but the detailed mechanism of their differentiation from osteoblasts remains unclear. The purpose of this study is to identify cell cycle regulators involved in the differentiation of osteoblasts into osteocytes and determine their physiological significance. The study uses IDG-SW3 cells as a model for the differentiation from osteoblasts to osteocytes. Among the major cyclin-dependent kinases (Cdks), Cdk1 is most abundantly expressed in IDG-SW3 cells, and its expression is down-regulated during differentiation into osteocytes. Inhibition of CDK1 activity reduces IDG-SW3 cell proliferation and differentiation into osteocytes. Osteocyte and Osteoblast-specific Cdk1 knockout in mice (Dmp1-Cdk1KO ) results in trabecular bone loss. Pthlh expression increases during differentiation, but inhibiting CDK1 activity reduces Pthlh expression. Parathyroid hormone-related protein concentration is reduced in the bone marrow of Dmp1-Cdk1KO mice. Four weeks of Parathyroid hormone administration partially recovers the trabecular bone loss in Dmp1-Cdk1KO mice. These results demonstrate that Cdk1 plays an essential role in the differentiation from osteoblast to osteocyte and the acquisition and maintenance of bone mass. The findings contribute to a better understanding of the mechanisms of bone mass regulation and can help develop efficient therapeutic strategies for osteoporosis treatment.

BACKGROUND: Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization.
METHODS: Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques.
RESULTS: After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells.
CONCLUSIONS: DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

Dentin matrix protein 1 (Dmp1) is a highly phosphorylated, extracellular matrix protein that is extensively expressed in bone and teeth but also found in soft tissues, including brain and muscle. However, the functions of Dmp1 in the mice cochlea are unknown. Our study showed that Dmp1 was expressed in auditory hair cells (HCs), with the role of Dmp1 in those cells identified using Dmp1 cKD mice. Immunostaining and scanning electron microscopy of the cochlea at P1 revealed that Dmp1 deficiency in mice resulted in an abnormal stereociliary bundle morphology and the mispositioning of the kinocilium. The following experiments further demonstrated that the cell-intrinsic polarity of HCs was affected without apparent effect on the tissue planer polarity, based on the observation that the asymmetric distribution of Vangl2 was unchanged whereas the Gαi3 expression domain was enlarged and Par6b expression was slightly altered. Then, the possible molecular mechanisms of Dmp1 involvement in inner ear development were explored via RNA-seq analysis. The study suggested that the Fgf23-Klotho endocrine axis may play a novel role in the inner ear and Dmp1 may regulate the kinocilium-stereocilia interaction via Fgf23-Klotho signaling. Together, our results proved the critical role of Dmp1 in the precise regulation of hair bundle morphogenesis in the early development of HCs.

BACKGROUND: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost.
METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene.
RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells.
CONCLUSIONS: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

The purpose of this study was to investigate whether fibroblast growth factor 4 (FGF4) and FGF9 are active in dentin differentiation. Dentin matrix protein 1 (Dmp1) -2A-Cre transgenic mice, which express the Cre-recombinase in Dmp1-expressing cells, were crossed with CAG-tdTomato mice as reporter mouse. The cell proliferation and tdTomato expressions were observed. The mesenchymal cell separated from neonatal molar tooth germ were cultured with or without FGF4, FGF9, and with or without their inhibitors ferulic acid and infigratinib (BGJ398) for 21 days. Their phenotypes were evaluated by cell count, flow cytometry, and real-time PCR. Immunohistochemistry for FGFR1, 2, and 3 expression and the expression of DMP1 were performed. FGF4 treatment of mesenchymal cells obtained promoted the expression of all odontoblast markers. FGF9 failed to enhance dentin sialophosphoprotein (Dspp) expression levels. Runt-related transcription factor 2 (Runx2) was upregulated until day 14 but was downregulated on day 21. Compared to Dmp1-negative cells, Dmp1-positive cells expressed higher levels of all odontoblast markers, except for Runx2. Simultaneous treatment with FGF4 and FGF9 had a synergistic effect on odontoblast differentiation, suggesting that they may play a role in odontoblast maturation.

Despite numerous studies on various surface modifications on titanium and its alloys, it remains unclear what kind of titanium-based surface modifications are capable of controlling cell activity. This study aimed to understand the mechanism at the cellular and molecular levels and investigate the in vitro response of osteoblastic MC3T3-E1 cultured on the Ti-6Al-4V surface modified by plasma electrolytic oxidation (PEO) treatment. A Ti-6Al-4V surface was prepared by PEO at 180, 280, and 380 V for 3 or 10 min in an electrolyte containing Ca2+/Pi ions. Our results showed that PEO-treated Ti-6Al-4V-Ca2+/Pi surfaces enhanced the cell attachment and differentiation of MC3T3-E1 compared to the untreated Ti-6Al-4V control but did not affect cytotoxicity as shown by cell proliferation and cell death. Interestingly, on the Ti-6Al-4V-Ca2+/Pi surface treated by PEO at 280 V for 3 or 10 min, MC3T3-E1 showed a higher initial adhesion and mineralization. In addition, the alkaline phosphatase (ALP) activity significantly increased in MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi (280 V for 3 or 10 min). In RNA-seq analysis, the expression of dentin matrix protein 1 (DMP1), sortilin 1 (Sort1), signal-induced proliferation-associated 1 like 2 (SIPA1L2), and interferon-induced transmembrane protein 5 (IFITM5) was induced during the osteogenic differentiation of MC3T3-E1 on the PEO-treated Ti-6Al-4V-Ca2+/Pi. DMP1 and IFITM5 silencing decreased the expression of bone differentiation-related mRNAs and proteins and ALP activity in MC3T3-E1. These results suggest that the PEO-treated Ti-6Al-4V-Ca2+/Pi surface induces osteoblast differentiation by regulating the expression of DMP1 and IFITM5. Therefore, surface microstructure modification through PEO coatings with Ca2+/Pi ions could be used as a valuable method to improve biocompatibility properties of titanium alloys.

The Dmp1-Cre mouse, expressing Cre from an 8-kb DNA fragment of the mouse Dmp1 gene, is a common tool to study gene functions in osteocytes. Here we report that the deletion of Tsc1 (TSC complex subunit 1) by 8 kb Dmp1-Cre causes rectal prolapse in mice. Histological examination shows the presence of colon polyps in Tsc1-deficient mice in association with significantly larger colon and narrower lumen, which recapitulates the common polyps pathology in Tuberous Sclerosis, an autosomal dominant disorder caused by mutations in either TSC1 or TSC2. The intestine in Tsc1-deficient mice is also enlarged with the presence of taller villi. Using the Ai14 reporter mice that express a red fluorescence protein upon Cre recombination, we show that 8 kb Dmp1-Cre activity is evident in portion of the mesenchyme of the colon and small intestine. Lastly, our data show that Tsc1 deletion by Dmp1-Cre leads to an increased proliferation in the mesenchyme of colon, which at least partly contributes to the polyps pathology seen in this mouse model and is likely a contributing factor of the polyps in Tuberous Sclerosis.

Abnormalities in calcium, phosphorus, PTH, vitamin D metabolism, bone, and vascular calcification occur in chronic kidney disease mineral bone disorder (CKD-MBD). Calciphylaxis, involving painful, ulcerative skin lesions, is also a major problem associated with CKD-MBD. There are no quality medical interventions to address these clinical issues. Bone ASARM peptides are strong inhibitors of mineralization and induce hypophosphatemia by inhibiting phosphate uptake from the gut. We hypothesize treatment of CKD-MBD rats with ASARM peptides will reverse hyperphosphatemia, reduce soft-tissue calcification, and prevent calciphylaxis.
To test our hypothesis, we assessed the effects of synthetic ASARM peptide in rats that had undergone a subtotal 5/6th nephrectomy (56NEPHREX), a rodent model of CKD-MBD. All rats were fed a high phosphate diet (2% Pi) to worsen mineral metabolism defects. Changes in serum potassium, phosphate, BUN, creatinine, PTH, FGF23, and calcium were assessed in response to 28 days of ASARM peptide infusion. Also, changes in bone quality, soft-tissue calcification, and expression of gut Npt2b (Slc34a2) were studied following ASARM peptide treatment.
Rats that had undergone 56NEPHREX treated with ASARM peptide showed major improvements in hyperphosphatemia, blood urea nitrogen (BUN), and bone quality compared with vehicle controls. Also, ASARM-infused 56NEPHREX rats displayed improved renal, brain, and cardiovascular calcification. Notably, ASARM peptide infusion prevented the genesis of subdermal medial blood vessel calcification and calciphylaxis-like lesions in 56NEPHREX rats compared with vehicle controls.
ASARM peptide infusion corrects hyperphosphatemia and improves vascular calcification, renal calcification, brain calcification, bone quality, renal function, and skin mineralization abnormalities in 56NEPHREX rats. These findings confirm our hypothesis and support the utility of ASARM peptide treatment in patients with CKD-MBD.