DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis.

Hypophosphatemia is a genetically heterogeneous disease. Here, we mapped an autosomal recessive form (designated ARHP) to chromosome 4q21 and identified homozygous mutations in DMP1 (dentin matrix protein 1), which encodes a non-collagenous bone matrix protein expressed in osteoblasts and osteocytes. Intact plasma levels of the phosphaturic protein FGF23 were clearly elevated in two of four affected individuals, providing a possible explanation for the phosphaturia and inappropriately normal 1,25(OH)2D levels and suggesting that DMP1 may regulate FGF23 expression.

Epileptic seizures and abnormal tooth development as primary presentation of pseudohypoparathyroidism type 1B.

Pseudohypoparathyroidism (PHP) is a rare genetic disorder characterised by a non-functioning PTH. Usually, the diagnosis is made following (symptomatic) hypocalcaemia. We describe a case in which epileptic seizures and abnormalities in dental development were the main clinical manifestation of PHP type 1B. This case demonstrates the importance of screening for hypocalcaemia in patients with de novo epileptic seizures. In addition, antiepileptic medications themselves may interfere with calcium-phosphate metabolism, causing or aggravating a hypocalcaemia as well. By correcting the calcium level, a resolution of these symptoms could be obtained.

Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23.

In contrast to the regulation of calcium homeostasis, which has been extensively studied over the past several decades, relatively little is known about the regulation of phosphate homeostasis. Fibroblast growth factor 23 (FGF23) is part of a previously unrecognized hormonal bone-parathyroid-kidney axis, which is modulated by PTH, 1,25(OH)(2)-vitamin D (1,25(OH)(2)D), dietary and serum phosphorus levels. Synthesis and secretion of FGF23 by osteocytes are positively regulated by 1,25(OH)(2)D and serum phosphorus and negatively regulated, through yet unknown mechanisms, by the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and by dentin matrix protein 1 (DMP1). In turn, FGF23 inhibits the synthesis of 1,25(OH)(2)D, and it may negatively regulate the secretion of parathyroid hormone (PTH) from the parathyroid glands. However, FGF23 synergizes with PTH to increase renal phosphate excretion by reducing expression of the renal sodium-phosphate cotransporters NaPi-IIa and NaPi-IIc in the proximal tubules. Most insights gained into the regulation of phosphate homeostasis by these factors are derived from human genetic disorders and genetically engineered mice, which are reviewed in this paper.

FGF23 and syndromes of abnormal renal phosphate handling.

Fibroblast growth factor 23 (FGF23) is part of a previously unrecognized hormonal bone-parathyroid-kidney axis, which is modulated by 1,25(OH)(2)-vitamin D (1,25(OH)(2)D), dietary and circulating phosphate and possibly PTH. FGF23 was discovered as the humoral factor in tumors that causes hypophosphatemia and osteomalacia and through the identification of a mutant form of FGF23 that leads to autosomal dominant hypophosphatemic rickets (ADHR), a rare genetic disorder. FGF23 appears to be mainly secreted by osteocytes where its expression is up-regulated by 1,25(OH)(2)D and probably by increased serum phosphate levels. Its synthesis and secretion is reduced through yet unknown mechanisms that involve the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Consequently, loss-of-function mutations in these genes underlie hypophosphatemic disorders that are either X-linked or autosomal recessive. Impaired O-glycosylation of FGF23 due to the lack of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3 (GALNT3) or due to certain homozygous FGF23 mutations results in reduced secretion of intact FGF23 and leads to familial hyperphosphatemic tumoral calcinosis. FGF23 acts through FGF-receptors and the coreceptor Klotho to reduce 1,25(OH)(2)D synthesis in the kidney and probably the synthesis of parathyroid hormone (PTH) by the parathyroid glands. It furthermore synergizes with PTH to increase renal phosphate excretion by reducing expression of the sodium-phosphate cotransporters NaPi-IIa and NaPi-IIc in the proximal tubules. Loss-of-function mutations in these two transporters lead to autosomal recessive Fanconi syndrome or to hereditary hypophosphatemic rickets with hypercalciuria, respectively.

Functional Properties of Two Distinct PTH1R Mutants Associated With Either Skeletal Defects or Pseudohypoparathyroidism.

Consistent with a vital role of parathyroid hormone (PTH) receptor type 1 (PTH1R) in skeletal development, homozygous loss-of-function PTH1R mutations in humans results in neonatal lethality (Blomstrand chondrodysplasia), whereas such heterozygous mutations cause a primary failure of tooth eruption (PFE). Despite a key role of PTH1R in calcium and phosphate homeostasis, blood mineral ion levels are not altered in such cases of PFE. Recently, two nonlethal homozygous PTH1R mutations were identified in two unrelated families in which affected members exhibit either dental and skeletal abnormalities (PTH1R-V204E) or hypocalcemia and hyperphosphatemia (PTH1R-R186H). Arg186 and Val204 map to the first transmembrane helix of the PTH1R, and thus to a critical region of this class B G protein-coupled receptor. We used cell-based assays and PTH and PTH-related protein (PTHrP) ligand analogs to assess the impact of the R186H and V204E mutations on PTH1R function in vitro. In transiently transfected HEK293 cells, PTH1R-R186H mediated cyclic adenosine monophosphate (cAMP) responses to PTH(1-34) and PTHrP(1-36) that were of comparable potency to those observed on wild-type PTH1R (PTH1R-WT) (half maximal effective concentrations [EC50s] = 0.4nM to 1.2nM), whereas the response-maxima were significantly reduced for the PTH1R-V204E mutant (maximum effect [Emax] = 81%-77% of PTH1R-WT, p ≤ 0.004). Antibody binding to an extracellular hemagglutinin (HA) tag was comparable for PTH1R-R186H and PTH1R-WT, but was significantly reduced for PTH1R-V204E (maximum binding level [Bmax] = 44% ± 11% of PTH1R-WT, p = 0.002). The potency of cAMP signaling induced by a PTH(1-11) analog was reduced by ninefold and threefold, respectively, for PTH1R-R186H and PTH1R-V204E, relative to PTH1R-WT, and a PTH(1-15) radioligand analog that bound adequately to PTH1R-WT exhibited little or no specific binding to either mutant receptor. The data support a general decrease in PTH1R surface expression and/or function as a mechanism for PFE and a selective impairment in PTH ligand affinity as a potential PTH1R-mutation-based mechanism for pseudohypoparathyroidism. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Novel regulators of phosphate homeostasis and bone metabolism.

The regulation of phosphate homeostasis remains incompletely understood. Most insights into the underlying mechanisms were established by defining the molecular basis of different inherited disorders that are characterized by an abnormal regulation of phosphate homeostasis. Using this approach, three novel regulators were previously identified, namely PHEX (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome), fibroblast growth factor (FGF)-23 and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Other studies had revealed heterozygous mutations in the sodium phosphate co-transporter NaPi-IIa as the cause of hypophosphatemia associated with hypercalciuria and osteoporosis, and homozygous or compound heterozygous mutations in NaPi-IIc were shown to cause hereditary hypophosphatemic rickets with hypercalciuria. Recently, positional cloning approaches furthermore led to the identification of homozygous inactivating mutations in dentin matrix protein 1 (DMP1) as the cause of an autosomal recessive form of hypophosphatemia. Using different immunometric assays, intact and C-terminal FGF-23 levels were found to be elevated in patients with oncogenic osteomalacia, and the tumors responsible for this disease showed increased expression of FGF-23 mRNA. Intact and C-terminal FGF-23 levels are furthermore elevated in patients with X-linked hypophosphatemia. This disorder is caused by inactivating PHEX mutations suggesting that this endopeptidase is somehow, most likely indirectly, involved in the metabolism of intact FGF-23. FGF-23 levels were also found to be elevated in some patients with ARHP indicating that the lack of DMP1 up-regulates expression of this phosphaturic hormone. The concentration of C-terminal FGF-23, but not of intact FGF-23, is significantly elevated in two forms of tumoral calcinosis (TC). One form of TC is caused by homozygous inactivating GALNT3 mutations implying that the encoded enzyme, which is involved in the initiation of O-glycosylation, is important for preventing cleavage of FGF-23 into biologically inactive fragments. The second form of tumoral calcinosis is caused by different homozygous FGF-23 mutations that affect conserved serine residues that may undergo O-glycosylation by GALNT3; the lack of this post-translational modification leads to an abnormal processing of FGF-23 and increased secretion of C-terminal fragments. It remains unknown whether and how the different phosphate-regulating proteins interact with each other and it appears very likely that additional proteins are involved in this process. It also remains unclear whether the dramatically elevated FGF-23 levels in patients with different stages of chronic kidney disease affect bone metabolism, particularly the mineralization of newly formed osteoid.

Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1-34) Peptide Fragment.

Polyethylene glycol (PEG) addition can prolong the pharmacokinetic and pharmacodynamic actions of a bioactive peptide in vivo, in part by impeding rates of glomerular filtration. For parathyroid hormone (PTH) peptides, pegylation could help in exploring the actions of the hormone in the kidney; e.g., in dissecting the relative roles that filtered versus blood-borne PTH play in regulating phosphate transport. It could also lead to potential alternate forms of treatment for hypoparathyroidism. We thus synthesized the fluorescent pegylated PTH derivative [Lys13 (tetramethylrhodamine {TMR}), Cys35 (PEG-20,000 Da)]PTH(1-35) (PEG-PTHTMR ) and its non-pegylated counterpart [Lys13 (TMR), Cys35 ]PTH(1-35) (PTHTMR ) and assessed their properties in cells and in mice. In PTHR1-expressing HEK-293 cells, PEG-PTHTMR and PTHTMR exhibited similar potencies for inducing cAMP signaling, whereas when injected into mice, the pegylated analog persisted much longer in the circulation (>24 hours versus ∼ 1 hour) and induced markedly more prolonged calcemic and phosphaturic responses than did the non-pegylated control. Fluorescence microscopy analysis of kidney sections obtained from the injected mice revealed much less PEG-PTHTMR than PTHTMR on the luminal brush-border surfaces of renal proximal tubule cells (PTCs), on which PTH regulates phosphate transporter function, whereas immunostained phosphorylated PKA substrate, a marker of cAMP signaling, was increased to similar extents for the two ligands and for each, was localized to the basolateral portion of the PTCs. Pegylation of a bioactive PTH peptide thus led to prolonged pharmacokinetic/pharmacodynamic properties in vivo, as well as to new in vivo data that support a prominent role for PTH action at basolateral surfaces of renal proximal tubule cells. © 2016 American Society for Bone and Mineral Research.

Osteocytic protein expression response to doxercalciferol therapy in pediatric dialysis patients.

BACKGROUND: Osteocytic protein expression is dysregulated in CKD and is affected by changes in mineral metabolism; however the effects of active vitamin D sterol therapy on osteocyte protein expression in advanced CKD is unknown. METHODS: Eleven pediatric patients with end stage kidney disease underwent bone biopsy, were treated for 8 months with doxercalciferol, and then underwent a second bone biopsy. Bone expression of fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP1), and sclerostin were determined by immunohistochemistry and quantified by Ariol Scanning. Western blot analysis and qRT-PCR was performed on bone abstracts of a subset of study subjects to determine the nature (i.e. size) of FGF23 and DMP1 in bone before and after therapy. RESULTS: As assessed by immunohistochemistry, bone FGF23, DMP1 and sclerostin protein all increased with therapy. In the case of FGF23, this increase was due to an increase in the full-length molecule without the appearance of FGF23 fragments. DMP1 was present primarily in its full-length form in healthy controls while 57kDa and 37kDa fragments of DMP1 were apparent in bone of dialysis patients at baseline and the 57 kDa appeared to decrease with therapy. CONCLUSION: Marked changes in osteocytic protein expression accompany doxercalciferol therapy, potentially impacting bone mineralization and the skeletal response to PTH. The effects of these bone changes on long-term outcomes remain to be determined.

Insights from genetic disorders of phosphate homeostasis.

The molecular identification and characterization of genetic defects leading to a number of rare inherited or acquired disorders affecting phosphate homeostasis has added tremendous detail to our understanding of the regulation of phosphate balance. The identification of the key phosphate-regulating hormone, fibroblast growth factor 23 (FGF23), as well as other molecules that control its production, such as the N-acetylgalactosaminyltransferase 3 GALNT3, the endopeptidase phosphate-regulating protein with homologies to endopeptidases on the X chromosome, and the matrix protein dentin matrix protein 1, and molecules that function as downstream effectors of FGF23, such as the longevity factor Klotho and the phosphate transporters NPT2a and NPT2c, has permitted us to understand the elegant and complex interplay that exists between the kidneys, bone, parathyroid, and gut. Such insights from genetic disorders have allowed not only the design of potent targeted therapies for some of these rare genetic disorders, such as using anti-FGF23 antibodies for treatment of X-linked hypophosphatemic rickets, but also have led to clinically relevant observations related to the dysregulation of mineral ion homeostasis in chronic kidney disease. Thus, we are able to leverage our knowledge of rare human disorders affecting only a few individuals, to understand and potentially treat disease processes that affect millions of patients.

Identification of a novel dentin matrix protein-1 (DMP-1) mutation and dental anomalies in a kindred with autosomal recessive hypophosphatemia.

An autosomal recessive form of hypophosphatemia (ARHP) was recently shown to be caused by homozygous mutations in DMP1, the gene encoding dentin matrix protein-1 (DMP-1), a non-collagenous bone matrix protein with an important role in the development and mineralization of bone and teeth. Here, we describe a previously not reported consanguineous ARHP kindred in which the three affected individuals carry a novel homozygous DMP-1 mutation. The index case presented at the age of 3 years with bowing of his legs and showed hypophosphatemia due to insufficient renal phosphate retention. Serum alkaline phosphatase activity was elevated, with initially normal PTH. FGF23 was inappropriately normal at an older age while being treated with oral phosphate and 1,25(OH)(2)D. Similar clinical and biochemical findings, except for elevated FGF23 levels, were present in his 16-month-old brother and his 12.5-year-old female cousin; the parents of the three affected children are first-degree cousins. Nucleotide sequence analysis was performed on PCR-amplified exons encoding DMP-1 and flanking intronic regions. A novel homozygous frame-shift mutation (c.485Tdel; p.Glu163ArgfsX53) in exon 6 resulting in a premature stop codon was identified in all effected individuals. The parents and available unaffected siblings were heterozygous for c.485Tdel. Tooth growth and shape were normal for the index case, his affected brother and cousin, but their permanent and deciduous teeth displayed enlarged pulp chambers. The identified genetic mutation underscores the importance of DMP-1 mutations in the pathogenesis of ARHP. Furthermore, DMP-1 mutations appear to contribute, through yet unknown mechanisms, to tooth development.

Long-term clinical outcome and carrier phenotype in autosomal recessive hypophosphatemia caused by a novel DMP1 mutation.

Homozygous inactivating mutations in DMP1 (dentin matrix protein 1), the gene encoding a noncollagenous bone matrix protein expressed in osteoblasts and osteocytes, cause autosomal recessive hypophosphatemia (ARHP). Herein we describe a family with ARHP owing to a novel homozygous DMP1 mutation and provide a detailed description of the associated skeletal dysplasia and carrier phenotype. The two adult patients with ARHP, a 78-year-old man and his 66-year-old sister, have suffered from bone pain and lower extremity varus deformities since early childhood. With increasing age, both patients developed severe joint pain, contractures, and complete immobilization of the spine. Radiographs showed short and deformed long bones, significant cranial hyperostosis, enthesopathies, and calcifications of the paraspinal ligaments. Biochemistries were consistent with hypophosphatemia owing to renal phosphate wasting; markers of bone turnover and serum fibroblast growth factor 23 (FGF-23) levels were increased significantly. Nucleotide sequence analysis of DMP1 revealed a novel homozygous mutation at the splice acceptor junction of exon 6 (IVS5-1G > A). Two heterozygous carriers of the mutation also showed mild hypophosphatemia, and bone biopsy in one of these individuals showed focal areas of osteomalacia. In bone, DMP1 expression was absent in the homozygote but normal in the heterozygote, whereas FGF-23 expression was increased in both subjects but higher in the ARHP patient. The clinical and laboratory observations in this family confirm that DMP1 has an important role in normal skeletal development and mineral homeostasis. The skeletal phenotype in ARHP may be significantly more severe than in other forms of hypophosphatemic rickets.

Disorders of phosphate homeostasis and tissue mineralisation.

Phosphate is absorbed from the diet in the gut, stored as hydroxyapatite in the skeleton, and excreted with the urine. The balance between these compartments determines the circulating phosphate concentration. Fibroblast growth factor 23 (FGF23) has recently been discovered and is part of a previously unrecognised hormonal bone-kidney axis. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome, and dentin matrix protein 1 regulate the expression of FGF23 in osteocytes, which then is O-glycosylated by UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase 3 and secreted into the circulation. FGF23 binds with high affinity to fibroblast growth factor receptor 1c in the presence of its co-receptor Klotho. It inhibits, either directly or indirectly, reabsorption of phosphate and the synthesis of 1,25-dihydroxy-vitamin-D by the renal proximal tubule and the secretion of parathyroid hormone by the parathyroid glands. Acquired or inborn errors affecting this newly discovered hormonal system can lead to abnormal phosphate homeostasis and/or tissue mineralisation. This chapter will provide an update on the current knowledge of the pathophysiology, the clinical presentation, diagnostic evaluation and therapy of the disorders of phosphate homeostasis and tissue mineralisation.

Patterns of FGF-23, DMP1, and MEPE expression in patients with chronic kidney disease.

Fibroblast growth factor 23 (FGF-23), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) are skeletal proteins involved in the regulation of phosphate homeostasis and bone metabolism. Circulating FGF-23 levels are increased in patients with chronic kidney disease (CKD); however, FGF-23 skeletal expression and its regulation by DMP1 and MEPE have yet to be evaluated. Thus, expression of these three proteins was characterized by immunohistochemistry in 32 pediatric and young adult patients with CKD stages 2-5. When compared to normal controls, bone FGF-23 and DMP1 expression were increased in all stages of CKD; significant differences in bone FGF-23 and DMP1 expression were not detected between pre-dialysis CKD and dialysis patients. Bone MEPE expression in CKD did not differ from controls. FGF-23 was expressed in osteocyte cell bodies located at the trabecular periphery. DMP1 was widely expressed in osteocyte cell bodies and dendrites throughout bone. MEPE was also expressed throughout bone, but only in osteocyte cell bodies. Bone FGF-23 expression correlated directly with plasma levels of the protein (r=0.43, p<0.01) and with bone DMP1 expression (r=0.54, p<0.01) and expression of both proteins were inversely related to osteoid accumulation. Bone MEPE expression was inversely related to bone volume. In conclusion, skeletal FGF-23 and DMP1 expression are increased in CKD and are related to skeletal mineralization. The patterns of expression of FGF-23, MEPE, and DMP1 differ markedly in trabecular bone, suggesting that individual osteocytes may have specialized functions. Increases in bone FGF-23 and DMP1 expression suggest that osteocyte function is altered early in the course of CKD.

Inherited hypophosphatemic disorders in children and the evolving mechanisms of phosphate regulation.

Phosphorous is essential for multiple cellular functions and constitutes an important mineral in bone. Hypophosphatemia in children leads to rickets resulting in abnormal growth and often skeletal deformities. Among various causes of low serum phosphorous are inherited disorders associated with increased urinary excretion of phosphate, including autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemia (XLH), autosomal recessive hypophosphatemia (ARHP), and hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Recent genetic analyses and subsequent biochemical and animal studies have revealed several novel molecules that appear to play key roles in the regulation of renal phosphate handling. These include a protein with abundant expression in bone, fibroblast growth factor 23 (FGF23), which has proven to be a circulating hormone that inhibits tubular reabsorption of phosphate in the kidney. Two other bone-specific proteins, PHEX and dentin matrix protein 1 (DMP1), appear to be necessary for limiting the expression of fibroblast growth factor 23, thereby allowing sufficient renal conservation of phosphate. This review focuses on the clinical, biochemical, and genetic features of inherited hypophosphatemic disorders, and presents the current understanding of hormonal and molecular mechanisms that govern phosphorous homeostasis.