Phosphate-regulating Endopeptidase Homolog X-linked Research Articles

Refractory rickets: A case of X-linked hypophosphatemic rickets (PHEX gene variation)

Hypophosphatemic rickets is a type of hereditary rickets distinguished by both hypophosphatemia and hyperphosphaturia. X-linked hypophosphatemic rickets (XLH) is the most prevalent type of heritable hypophosphatemic rickets. We describe a 3-year-old child whose phosphate-regulating endopeptidase homolog X-linked (PHEX) gene variation resulted in lower limb abnormalities and short stature. A similar history was noted in the father. We highlight the significance of a prompt diagnosis and initiation of treatment to prevent subsequent sequelae.

Experience of X-linked hypophosphatemic rickets in the Gulf Cooperation Council countries: case series.

X-linked hypophosphatemic rickets (XLH), the most prevalent form of inherited hypophosphatemic rickets, is caused by loss-of-function mutations in the gene encoding phosphate-regulating endopeptidase homolog, X-linked (PHEX). This case series presents 14 cases of XLH from Gulf Cooperation Council (GCC) countries. The patients' medical history, biochemical and radiological investigative findings, as well as treatment responses and side effects from both conventional and burosumab therapy, are described. Cases were aged 2-40 years at diagnosis. There were two male cases and 12 female cases. All cases were treated with conventional therapy which resulted in a lack of improvement in or worsening of the clinical signs and symptoms of rickets or biochemical parameters. Side effects of conventional therapy included nausea, diarrhea, abdominal pain, nephrocalcinosis, and hyperparathyroidism, which affected the patients' quality of life and adherence to treatment. In the 10 patients treated with burosumab, there was a marked improvement in the biochemical markers of rickets, with a mean increase in serum phosphate of +0.56 mmol/L and tubular maximum phosphate reabsorption (TmP) to glomerular filtration rate (GFR) ratio (TmP/GFR) of +0.39 mmol/L at 12 months compared to baseline. Furthermore, a mean decrease in serum alkaline phosphatase (ALP) of -80.80 IU/L and parathyroid hormone (PTH) of -63.61 pmol/L at 12 months compared to baseline was observed in these patients. Additionally, patients treated with burosumab reported reduced pain, muscle weakness, and fatigue as well as the ability to lead more physically active lives with no significant side effects of treatment. Conventional therapy resulted in a suboptimal response, with a lack of improvement of clinical signs and symptoms. Side effects of conventional therapy included nausea, diarrhea, abdominal pain, nephrocalcinosis, and hyperparathyroidism, which affected the patients' quality of life and adherence to treatment. Burosumab demonstrated marked improvements in the biochemical markers of rickets, in addition to reducing pain, muscle weakness, and fatigue. There were no significant side effects associated with burosumab therapy.

Characterization of novel PHEX variants in X-linked hypophosphatemic rickets and genotype-PHEX activity correlation.

X-linked hypophosphatemia (XLHR) is the most common genetic form of hypophosphatemic rickets (HR), which is caused by phosphate regulating endopeptidase homolog X-linked (PHEX) gene mutation. At present, the genotype-phenotype relationship of XLHR and the pathogenic role of PHEX have not been fully understood. In this study, we summarized clinical features in a new cohort of 49 HR patients and detected 16 novel PHEX and 5 novel non-PHEX variants. Subsequently, we studied the pathogenesis of new variants by protein expression, glycosylation analysis, subcellular localization and endopeptidase activity. The results showed that missense variants (Q189H and X750R) slightly reduced protein expression without obviously altering protein length and localization, whereas truncating variants significantly impaired the synthesis of PHEX and produced a shorter immature protein in cells. Interestingly, no evident correlation was observed between mutation types and clinical phenotypes. However, when we analyzed the relationship between PHEX activity and serum phosphorus level, we found that patients with low PHEX activity tended to have severe hypophosphatemia and high rickets severity score (RSS). Following this observation, we established two new knock-in XLHR mouse models with two novel Phex variants (c.T1349C and c.C426G, respectively) using CRISPR/Cas9 technology. Both mouse models demonstrated clinical manifestations of XLHR seen in patients and PhexC426G mice showed more severe phenotype than PhexT1349C mice, which further confirmed the rationality of genotype-PHEX enzymatic activity correlation analysis. Therefore, our findings demonstrated that novel PHEX variants could disrupt protein function via affecting protein synthesis, post-translational modification, cellular trafficking and catalytic activity. Our study facilitates a better understanding of XLHR pathogenic mechanism and PHEX activity-phenotype correlation, which is of crucial importance for future diagnosis and treatment of XLHR.

A genetic study of a Brazilian cohort of patients with X-linked hypophosphatemia reveals no correlation between genotype and phenotype.

X-linked hypophosphatemia (XLH) is the most common inherited form of rickets, and it is caused by pathogenic inactivating variants of the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene. The main purpose of this study is to identify the presence of a genotype-phenotype correlation in a cohort of XLH patients. This is a retrospective study including patients diagnosed with hypophosphatemic rickets, confirmed by clinical, radiological, and laboratory findings. Medical records were reviewed for phenotypic analyses. Genomic DNA was extracted from the peripheral blood lymphocytes, and PHEX sequencing was performed by exomic NGS sequencing. The Wilcoxon rank-sum test and the two-tailed Fisher's exact test were employed for the statistical analyses of this study. A total of 41 patients were included in this study, and 63.41% (26/41) of the patients were female. The mutation analyses identified 29.27% missense variants and 29.72% nonsense variants, most of them were considered deleterious (66.41%). Six novel deleterious variants in the PHEX gene were detected in seven patients. The median concentrations of pretreatment serum calcium, phosphorus, and parathyroid hormone (PTH) were not significantly different among patients with different genotypes. An orthopedic surgery due to bone deformity was required in 57.69%. Our analysis did not identify any specific genotype as a predictor. No significant genotype-phenotype correlation was found, suggesting that the recognition of subjacent pathogenic mutation in the PHEX gene may have limited prognostic value. Despite this finding, genetic testing may be useful for identifying affected individuals early and providing appropriate treatment.

Differing responses of osteogenic cell lines to β-glycerophosphate

Ascorbic acid (Asc), dexamethasone (Dex) and β-glycerophosphate (β-Gly) are commonly used to promote osteogenic behaviour by osteoblasts in vitro. According to the literature, several osteosarcoma cells lines appear to respond differently to the latter with regards to proliferation kinetics and osteogenic gene transcription. Unsurprisingly, these differences lead to contrasting data between publications that necessitate preliminary studies to confirm the phenotype of the chosen osteosarcoma cell line in the presence of Asc, Dex and β-Gly. The present study exposed Saos-2 cells to different combinations of Asc, Dex and β-Gly for 14 days and compared the response with immortalised human mesenchymal stromal/stem cells (MSCs). Cell numbers, cytotoxicity, mineralised matrix deposition and cell proliferation were analysed to assess osteoblast-like behaviour in the presence of Asc, Dex and β-Gly. Additionally, gene expression of runt-related transcription factor 2 (RUNX2); osteocalcin (OCN); alkaline phosphatase (ALP); phosphate regulating endopeptidase homolog X-linked (PHEX); marker of proliferation MKI67 and proliferating cell nuclear antigen (PCNA) was performed every two days during the 14-day cultures. It was found that proliferation of Saos-2 cells was significantly decreased by the presence of β-Gly which contrasted with hMSCs where no change was observed. Furthermore, unlike hMSCs, Saos-2 cells demonstrated an upregulated expression of late osteoblastic markers, OCN and PHEX that suggested β-Gly could affect later stages of osteogenic differentiation. In summary, it is important to consider that β-Gly significantly affects key cell processes of Saos-2 when using it as an osteoblast-like cell model.

Impact of X-Linked Hypophosphatemia on Muscle Symptoms

X-linked hypophosphatemia (XLH) is the most common hereditary form of rickets and deficiency of renal tubular phosphate transport in humans. XLH is caused by the inactivation of mutations within the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene and follows an X-dominant transmission. It has an estimated frequency of 1 case per 20,000, and over 300 distinct pathogenic variations have been reported that result in an excess of fibroblast growth factor 23 (FGF23) in the serum. Increased levels of FGF23 lead to renal phosphate loss, decreased serum 1,25-dihydroxyvitamin D, and increased metabolism of 1,25-dihydoxyvitamin D, resulting in hypophosphatemia. Major clinical manifestations include rickets, bone deformities, and growth retardation that develop during childhood, and osteomalacia-related fractures or pseudo-fractures, degenerative osteoarthritis, enthesopathy, dental anomalies, and hearing loss during adulthood, which can affect quality of life. In addition, fatigue is also a common symptom in patients with XLH, who experience decreased motion, muscle weakness, and pain, contributing to altered quality of life. The clinical and biomedical characteristics of XLH are extensively defined in bone tissue since skeletal deformations and mineralization defects are the most evident effects of high FGF23 and low serum phosphate levels. However, despite the muscular symptoms that XLH causes, very few reports are available on the effects of FGF23 and phosphate in muscle tissue. Given the close relationship between bones and skeletal muscles, studying the effects of FGF23 and phosphate on muscle could provide additional opportunities to understand the interactions between these two important compartments of the body. By describing the current literature on XLH and skeletal muscle dysfunctions, the purpose of this review is to highlight future areas of research that could contribute to a better understanding of XLH muscular disability and its management.

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

Pathogenic Variants of the PHEX Gene

Twenty-five years ago, a pathogenic variant of the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene was identified as the cause of X-linked hypophosphatemic rickets (XLH). Subsequently, the overproduction of fibroblast growth factor 23 (FGF23) due to PHEX defects has been found to be associated with XLH pathophysiology. However, the mechanism by which PHEX deficiency contributes to the upregulation of FGF23 and the function of PHEX itself remain unclear. To date, over 700 pathogenic variants have been identified in patients with XLH, and functional assays and genotype–phenotype correlation analyses based on pathogenic variant data derived from XLH patients have been reported. Genetic testing for XLH is useful for the diagnosis. Not only have single-nucleotide variants causing missense, nonsense, and splicing variants and small deletion/insertion variants causing frameshift/non-frameshift alterations been observed, but also gross deletion/duplication variants causing copy number variants have been reported as pathogenic variants in PHEX. With the development of new technologies including next generation sequencing, it is expected that an increasing number of pathogenic variants will be identified. This chapter aimed to summarize the genotype of PHEX and related analyses and discusses the pathophysiology of PHEX defects to seek clues on unsolved questions.

Bone Deformities and Kidney Failure: Coincidence of PHEX-Related Hypophosphatemic Rickets and m.3243A>G Mitochondrial Disease.

X-linked hypophosphatemic rickets (XLH) and m.3243A>G mitochondrial disease share several clinical findings, including short stature, hearing impairment (HI), nephropathy, and hypertension. Here, we report on a case with the rare coincidence of these two genetic conditions. In early childhood, the patient presented with hypophosphatemia and bone deformities and was clinically diagnosed with XLH. This was genetically verified in adulthood with the identification of a de novo pathogenic deletion in phosphate-regulating endopeptidase homolog X-linked (PHEX). In addition, the patient developed HI and hypertension and when his mother was diagnosed with m.3243A>G, subsequent genetic testing confirmed the patient to carry the same variant. Over the next two decades, the patient developed progressive renal impairment however without nephrocalcinosis known to associate with XLH which could indicate an m.3243A>G-related kidney disease. Parallel with the progression of renal impairment, the patient developed hyperphosphatemia and secondary hyperparathyroidism. In conclusion, this case represents a complex clinical phenotype with the reversal of hypo- to hyperphosphatemia in XLH potentially mediated by the development of an m.3243A>G-associated nephropathy.

Genotype-phenotype analysis, and assessment of the importance of the zinc-binding site in PHEX in Japanese patients with X-linked hypophosphatemic rickets using 3D structure modeling.

X-linked hypophosphatemic rickets (XLH) is an inheritable type of rickets caused by inactivating variants in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene, which results in the overproduction of fibroblast growth factor 23 (FGF23). The mechanism by which PHEX impairment leads to FGF23 overproduction is unknown. Because little is known regarding the genotype-phenotype correlation in Japanese XLH, we summarized the available clinical and genetic data and analyzed the genotype-phenotype relationships using 3-dimensional (3D) structure modeling to clarify the XLH pathophysiology. We retrospectively reviewed the clinical features and performed genetic analysis of 39 Japanese patients with XLH from 28 unrelated pedigrees carrying any known or novel PHEX variant. To predict changes in the 3D structure of mutant PHEX, we constructed a putative 3D model of each mutant and evaluated the effect of structural alteration by genotype-phenotype correlation analysis. Genetic analysis revealed 23 PHEX variants, including eight novel variants. They were associated with high i-FGF23 levels, hypophosphatemia, phosphaturia, high alkaline phosphatase levels, and short stature. No gene dosage effect or genotype-phenotype correlation was observed when truncating and non-truncating variants were compared. However, the conservation of the zinc-binding site and cavity in PHEX had an impact on the elevation of i-FGF23 levels. Via genotype-phenotype relationship analysis using 3D modeling, we showed that the zinc-binding site and cavity in PHEX can play a critical role in its function. These findings provide new genetic clues for investigating the function of PHEX and the pathogenesis of XLH.

Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene

BackgroundIn the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene.ResultsIn this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX).ConclusionsOverall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.Graphic abstract

Long-term effect of conventional phosphate and calcitriol treatment on metabolic recovery and catch-up growth in children with PHEX mutation.

Hereditary hypophosphatemic rickets (HR) is conventionally treated with phosphate and calcitriol. Exploring genotype and phenotypic spectrum of X-linked hypophosphatemic rickets (XLHR), focusing on short-term, long-term, and pubertal impact of conventional treatment was aimed. Sixteen patients from 12 unrelated families with HR were analyzed for phosphate regulating endopeptidase homolog X-linked (PHEX) mutation. Initially Sanger sequencing analysis was performed. If PHEX mutation was not detected, multiplex ligation-dependent probe amplification (MLPA) was performed. If molecular defect was detected, first-degree relatives were analyzed. Thirteen patients (81%) and five first-degree relatives with XLHR were evaluated for genotype-phenotype or gender-phenotype correlation. Clinical characteristics and response to conventional treatment were determined retrospectively. Nine different PHEX mutations were identified; four splice-site, three point mutations, and two single exon deletions. Four were novel mutations. Despite conventional treatment, median adult height was lower than median height on admission (-3.8 and-2.3 SDS, respectively), metabolic and radiographic recovery were not achieved, adherence was low (30%). Although mean adult height was better in compliant patients than noncompliants (-2.6 vs.-3.7 SDS, respectively), they were still short. Correlation between phenotype and genotype or gender could not be shown. Median phosphate decreased significantly throughout puberty (p=0.014). Median pubertal height was lower than prepubertal height (-4.4 vs.-3.6 SDS; respectively), pubertal growth spurt was not observed. Among five patients with a follow-up longer than five years, three had nephrocalcinosis (60%), two had hyperparathyroidism (40%), 4/6 (33%) required correction osteotomy. Conventional treatment appears to have limited effect on metabolic, clinical and radiographic recovery in XLHR. Metabolic control and growth worsened during puberty. Although, long-term adverse effects are yet to be seen, introduction of burosumab as first-line treatment may be an alternative after infancy.

Diagnosis and management of X-linked hypophosphatemia in children and adolescent in the Gulf Cooperation Council countries

IntroductionX-linked hypophosphatemia (XLH) is a rare inherited cause of hypophosphatemic rickets and osteomalacia. It is caused by mutations in the phosphate-regulating endopeptidase homolog, X-linked (PHEX). This results in increased plasma fibroblast growth factor-23 (FGF23), which leads to loss of renal sodium-phosphate co-transporter expression leading to chronic renal phosphate excretion. It also leads to low serum 1,25-dihydroxyvitamin D (1,25(OH)2D), resulting in impaired intestinal phosphate absorption. Chronic hypophosphatemia in XLH leads to impaired endochondral mineralization of the growth plates of long bones with bony deformities. XLH in children and adolescents also causes impaired growth, myopathy, bone pain, and dental abscesses. XLH is the most frequent inherited cause of phosphopenic rickets/osteomalacia. Hypophosphatemia is also found in calcipenic rickets/osteomalacia as a result of secondary hyperparathyroidism. Thus, chronic hypophosphatemia is a common etiologic factor in all types of rickets.ResultsThere is considerable overlap between symptoms and signs of phosphopenic and calcipenic rickets/osteomalacia. Wrong diagnosis leads to inappropriate treatment of rickets/osteomalacia. Nutritional rickets and osteomalacia are common in the Gulf Cooperation Council countries which include Saudi Arabia, United Arab Emirates, Kuwait, Qatar, Bahrain, and Oman. Due to high levels of consanguinity in the region, genetic causes of phosphopenic and calcipenic rickets/osteomalacia are also common.ConclusionThis guideline was developed to provide an approach to the diagnosis of XLH, especially where there is no family history of the disease, and that other related conditions are not mistaken for XLH. We also guide the medical management of XLH with conventional treatment and with burosumab, a recombinant human IgG1 monoclonal antibody to FGF23.

X-Linked Hypophosphatemia: A New Era in Management.

X-linked hypophosphatemia (XLH) is a rare, hereditary, progressive musculoskeletal disease that often causes pain and short stature, as well as decreased physical function, mobility, and quality of life. Hypophosphatemia in XLH is caused by loss of function mutations in the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene, resulting in excess levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), which leads to renal phosphate wasting and decreased serum 1,25-dihydroxyvitamin D production. Historically, treatment options were limited to oral phosphate and active vitamin D analogues (conventional management) dosed several times daily in an attempt to improve skeletal mineralization by increasing serum phosphorus. The recent approval of burosumab, a fully human monoclonal antibody to FGF23, has provided a new, targeted treatment option for patients with XLH. This review summarizes our current understanding of XLH, the safety and efficacy of conventional management and burosumab, existing recommendations for managing patients, and unanswered questions in the field.

Development of Enthesopathies and Joint Structural Damage in a Murine Model of X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH) is characterized by rickets and osteomalacia, caused by inactivating mutations in the Phosphate-regulating endopeptidase homolog X-linked (PHEX) gene. With aging, adult patients develop paradoxical heterotopic calcifications of tendons and ligaments at their insertion sites (enthesophytes), and joint alterations. Understanding the progression of this structural damage that severely affects patients’ quality of life will help to improve the management of XLH. Here, we characterized the occurrence of enthesophytes and joint alterations through a 12 month in vivo micro-CT follow-up in the Hyp mouse, a murine model of XLH (n = 5 mice per group). Similar to adult patients with XLH, Hyp mice developed calcaneal enthesophytes, hip joint alterations, erosions of the sacroiliac joints and periarticular calcifications. These lesions were already present at month 3 and gradually worsened over time. In sharp contrast, no abnormalities were observed in control mice at early time points. Histological analyses confirmed the presence of bone erosions, calcifications and expansion of mineralizing enthesis fibrocartilage in Hyp mice and their absence in controls and suggested that new bone formation is driven by altered mechanical strain. Interestingly, despite a strong deformation of the curvature, none of the Hyp mice displayed enthesophyte at the spine. Peripheral enthesophytes and joint alterations develop at the early stages of the disease and gradually worsen overtime. Overall, our findings highlight the relevance of this preclinical model to test new therapies aiming to prevent bone and joint complications in XLH.

Active sites of human MEPE-ASARM regulating bone matrix mineralization

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.

Biological stenciling of mineralization in the skeleton: Local enzymatic removal of inhibitors in the extracellular matrix.

Biomineralization is remarkably diverse and provides myriad functions across many organismal systems. Biomineralization processes typically produce hardened, hierarchically organized structures usually having nanostructured mineral assemblies that are formed through inorganic-organic (usually protein) interactions. Calcium‑carbonate biomineral predominates in structures of small invertebrate organisms abundant in marine environments, particularly in shells (remarkably it is also found in the inner ear otoconia of vertebrates), whereas calcium-phosphate biomineral predominates in the skeletons and dentitions of both marine and terrestrial vertebrates, including humans. Reconciliation of the interplay between organic moieties and inorganic crystals in bones and teeth is a cornerstone of biomineralization research. Key molecular determinants of skeletal and dental mineralization have been identified in health and disease, and in pathologic ectopic calcification, ranging from small molecules such as pyrophosphate, to small membrane-bounded matrix vesicles shed from cells, and to noncollagenous extracellular matrix proteins such as osteopontin and their derived bioactive peptides. Beyond partly knowing the regulatory role of the direct actions of inhibitors on vertebrate mineralization, more recently the importance of their enzymatic removal from the extracellular matrix has become increasingly understood. Great progress has been made in deciphering the relationship between mineralization inhibitors and the enzymes that degrade them, and how adverse changes in this physiologic pathway (such as gene mutations causing disease) result in mineralization defects. Two examples of this are rare skeletal diseases having osteomalacia/odontomalacia (soft bones and teeth) - namely hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH) - where inactivating mutations occur in the gene for the enzymes tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, ALPL) and phosphate-regulating endopeptidase homolog X-linked (PHEX), respectively. Here, we review and provide a concept for how existing and new information now comes together to describe the dual nature of regulation of mineralization - through systemic mineral ion homeostasis involving circulating factors, coupled with molecular determinants operating at the local level in the extracellular matrix. For the local mineralization events in the extracellular matrix, we present a focused concept in skeletal mineralization biology called the Stenciling Principle - a principle (building upon seminal work by Neuman and Fleisch) describing how the action of enzymes to remove tissue-resident inhibitors defines with precision the location and progression of mineralization.

Functional Characterization of PHEX Gene Variants in Children With X-Linked Hypophosphatemic Rickets Shows No Evidence of Genotype-Phenotype Correlation.

X-linked hypophosphatemia (XLHR) is caused by loss-of-function mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Considerable controversy exists regarding genotype-phenotype correlations in XLHR. The present study describes the clinical features and molecular genetic bases of 53 pediatric patients with XLHR. Overall, 47 different mutations were identified, of which 27 were not previously described in the literature or entered in the Human Gene Mutation Database (HGMD). A high prevalence (72.34%) of truncating variants was observed in XLHR patients. The clinical presentation and severity of XLHR did not show an evident correlation between the truncating and non-truncating mutation types in our cohort. To further delineate the characteristics of PHEX variants underlying this nonsignificant trend, we assessed the effects of 10 PHEX variants on protein expression, cellular trafficking, and endopeptidase activity. Our results showed that the nonsense mutations p.Arg567*, p.Gln714*, and p.Arg747* caused a reduction of protein molecular weight and a trafficking defect. Among seven non-truncating mutations, the p.Cys77Tyr, p.Cys85Ser, p.Ile281Lys, p.Ile333del, p.Ala514Pro, and p.Gly572Ser mutants were not secreted into the medium and remained trapped inside cells in an immature form, whereas the p.Gly553Glu mutant was terminally glycosylated and secreted into the medium. We further assessed the endopeptidase activity of the p.Gly553Glu mutant using a quenched fluorogenic peptide substrate and revealed that the activity of p.Gly553Glu significantly reduced to 13% compared with the wild type, which indicated disruption of catalytic function. These data not only support the clinical results showing no correlation between disease severity and the type of PHEX mutation but also provide helpful molecular insights into the pathogenesis of XLHR. © 2020 American Society for Bone and Mineral Research.

Predicting the Impact of PHEX, FGF23 and DMP1 Gene Variants Found in Malaysian Malay Patients with Hypophosphataemic Rickets Through In Silico Analysis of Protein Function and mRNA Secondary Structure

Hypophosphataemic Rickets (HR) is a rare bone disorder characterised by chronic hypophosphataemia caused by defective phosphate reabsorption in the renal tubules. Variants in phosphate-regulating endopeptidase homolog, X-linked (PHEX), fibroblast growth factor-23 (FGF23) and dentin matrix protein-1 (DMP1) genes contribute to X-linked dominant, autosomal dominant and autosomal recessive forms of HR, respectively. In this study, four Malaysian patients’ DNA samples were subjected to polymerase chain reaction and Sanger sequencing to identify the types and locations of the variants. Then, in silico study was conducted based on the variants found to predict the effects of amino acid substitution on protein functions using SIFT and PolyPhen-2 software and RNAfold was used to construct the mRNA secondary structure. Mutational analyses had revealed two variants in PHEX; c.10G>C (E4Q), c.1970A>G (Y657C), one mutation in FGF23; c.716C>T (T239M) and three variants on DMP1; c.309A>T (S69C), c.1322C>T (S406S), c.1334G>A (E410E). The variants in these Malay patients were previously reported in different ethnic HR patients. Protein prediction programs suggested that the PHEX Y657C and DMP1 S69C variants may affect protein function. All variants were predicted to alter the secondary mRNA structure. These findings suggest that these missense and silent variants may lead to changes in protein function and mRNA secondary structure that are associated with the manifestation of HR phenotype.

Primary Human Osteocyte Networks in Pure and Modified Collagen Gels.

Osteocytes play a key role in orchestrating bone homeostasis and turnover and, therefore, in vitro investigations with osteocytes are of high relevance for biomaterial and drug testing in future. In this study, collagen type I gels and collagen gels modified with biomimetically mineralized collagen were tested as three-dimensional (3D) environment for the maintenance of the osteocytic phenotype of primary human osteocytes. After cultivation in different collagen gels, cells were analyzed microscopically for the osteocytic phenotype and gene expression of osteocyte marker genes osteocalcin, podoplanin (PDPN)/E11, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP-1), and sclerostin (SOST). Directly after isolation from bone tissue the cells expressed all examined osteocyte markers. After 7 days of 3D cultivation in collagen gels the osteocytic marker genes MEPE and SOST were upregulated and the other marker genes still expressed. Modification of collagen gels with biomimetically mineralized collagen and strontium-doped mineralized collagen prevented the cell-seeded gels from shrinking. Osteocyte morphology was not affected by the gel modification. However, the isolation of RNA from the mineralized gel variants was heavily impaired. Alternatively, the osteocytic differentiation of human osteoblasts in the different collagen gels was examined. Primary human osteoblasts were embedded into the gels and cultivated under osteogenic stimulation. After 14 days of cultivation, embedded osteoblasts showed osteocyte-like morphology and positive staining for DMP-1. Early osteocyte marker genes, such as PDPN/E11 and PHEX, were expressed while the expression of the osteoblast marker gene alkaline phosphatase (ALPL) increased. This ALPL upregulation was partly prevented by modification of collagen gels with mineralized collagen. Impact Statement This research focuses on the in vitro three-dimensional cultivation of primary human osteocytes instead of rodent osteocyte cell lines. Stable in vitro cultures of these regulating cells provide the opportunity to establish co- and triple cultures with osteoblasts and osteoclasts to analyze the cross talk between these cell species and to establish in vitro bone models for the testing of bioactive molecules, growth factors, drugs, and biomaterials.