Defective Bone Mineralization Research Articles

Hypophosphatasia - Recent Advances in Diagnosis and Treatment

Hypophosphatasia (HP) is a rare inborn error of bone and mineral metabolism transmitted as an autosomal- recessive trait. It is characterized by a reduced activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSAP) and elevated concentrations of its substrates including pyrophosphates. Clinical symptoms include defective bone mineralisation with bone deformities, fractures and recently defined chronic non-bacterial osteomyelitis. Renal damage due to calcification, dental abnormalities with premature loss of dentition and craniosynostosis are further symptoms. Knowledge on the mechanisms underlying cell activation leading to inflammation and tissue destruction is still limited in HP. Recent investigations have provided evidence that calcium pyrophosphate crystals are essentially involved in activating inflammatory signal transduction pathways via different receptors of the innate immune system. Further studies are needed to improve our understanding of the pathophysiological mechanisms leading to inflammation and tissue destruction associated with deposition of microcrystals. They might support the development of new therapeutic strategies for crystal-induced inflammation. Laboratory assays, genetic analysis and radiographic imaging can confirm the diagnosis. Since clinical symptoms are highly variable patients should be followed by a HP-experienced multidisciplinary team (paediatrician, radiologist, orthopedic surgeon, neurosurgeon, dentist, nutritional specialist). At the moment symptomatic treatment is most important because curative or causative therapies, like gene transfer or enzyme replacement therapy, are not yet available.

GPRC6A Null Mice Exhibit Osteopenia, Feminization and Metabolic Syndrome

BackgroundGPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.Methods/Principal FindingsIn this study, we created and characterized the phenotype of GPRC6A −/− mice. We observed complex metabolic abnormalities in GPRC6A −/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A −/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A −/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A −/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.Conclusions/Significance GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Intramedullary Nailing for Adult Hypophosphatasia: A Case Report

Hypophosphatasia is a rare genetic metabolic disorder characterised by defective bone mineralisation secondary to serum and bone alkaline phosphatase deficiency. We report a 46-year-old woman who underwent multiple intramedullary nailings for fractures and deformities of 6 long bones over 13 years.

Hypophosphatasia may lead to bone fragility: don’t miss it

Hypophosphatasia is an inheritable disorder characterised by defective bone mineralisation due to the impaired activity of tissue-non-specific alkaline phosphatase (AP). Clinical presentation ranges from stillbirth without mineralised bone to pathological fractures in late adulthood. During childhood, the main manifestations include rickets, growth delay and dental problems. Fractures and bone pain usually characterise the adult form. A 9-year-old girl was referred for repetitive fractures after minimal trauma. She had normal growth, normal sclerae, no rickets and minimal dental abnormalities. Her sister had also presented fractures. The proband, her sister and mother had low total and bone-specific AP levels and E435K mutation in exon 12 of the liver/bone/kidney AP gene. Low AP levels must lead to genetic analysis. Bone fragility and repetitive fractures may be symptoms of hypophosphatasia in childhood, which must not be neglected. Associated factors such as vitamin D or calcium deficiency must be prevented. In conclusion, hypophosphatasia must not be forgotten as an aetiological factor of repetitive fractures or bone pain in children and AP activity should be checked accurately.

Gastrointestinale Erkrankungen und Osteomalazie

Osteomalacia is a metabolic bone disease associated with impaired mineralization of the bone due to Vitamin D and Calcium deficiency that can develop in gastrointestinal disorders. Gastrointestinal malabsorption after surgery, in disorders of the small bowl, in diseases of the hepatobiliary tree and in pancreatic insufficiency can lead to decreased enteral resorption of the fat-soluble Vitamin D and/or depletion of endogenous Vitamin D stores due to abnormal enterohepatic circulation. As a consequence of the Vitamin D deficiency in combination with the underlying condition patients develop an impaired calcium absorption resulting in hypocalcaemia, which leads to defective bone mineralization. Additionally chronic gastrointestinal inflammation and corticosteroid therapy - which is often needed in these patients - have a negative effect on bone metabolism as well. The therapy consists of oral substitution of Vitamin D and Calcium as well as sufficient sun light exposure or in severe cases the use of artificial UVB-radiation.

Does FGF23 toxicity influence the outcome of chronic kidney disease?

Maintenance of physiologic phosphate balance is important for essential cellular functions [1]. Dysregulation of the phosphate balance in the form of hypophosphataemia can lead to the development of myopathy, cardiac dysfunction, haematological abnormalities and bone mineralization defects [1]. In contrast, hyperphosphataemia can cause vascular and soft tissue calcification [2,3]. Studies have convincingly demonstrated that FGF23 is a master regulator of systemic phosphate homeostasis [4–9]. FGF23 is a 30 kDa protein that is proteolytically processed to generate smaller N-terminal (∼18 kDa) and Cterminal (∼12 kDa) fragments. The N-terminal fragment of FGF23 contains the FGF receptor-binding domain, while the C-terminal fragment is proposed to be necessary for interaction with Klotho (a type 1 membrane protein with homology to s-glucosidase), which is believed to be a cofactor in FGF23–FGF receptor interactions [10]. FGF23 is a circulating phosphaturic factor that controls systemic phosphate homeostasis by regulating renal inorganic phosphate reabsorption [5]. The expression of members of the sodium phosphate co-transporter family (Na/Pi-2a and Na/Pi-2c) that mediate phosphate uptake in proximal tubular epithelial cells can be suppressed by FGF23 [11]. By suppressing Na/Pi co-transporter activity, FGF23 can reduce renal phosphate reabsorption, thereby increasing urinary phosphate excretion. The in vivo phosphaturic effect of FGF23 is convincingly demonstrated in animal studies. For instance, transgenic mice overexpressing human or mouse FGF23/Fgf23 have severe urinary phosphate wasting due to the suppression of renal Na/Pi co-transporter activity [12–14]. Inactivation of Fgf23 function in mice resulted

Infantile Hypophosphatasia due to a New Compound Heterozygous TNSALP Mutation – Functional Evidence for a Hydrophobic Side-Chain?

Infantile hypophosphatasia (IH) is an inherited disorder characterized by defective bone mineralization and a deficiency of alkaline phosphatase activity. The aim of the study was to evaluate a new compound heterozygous TNSALP mutation for its residual enzyme activity and localization of the comprised amino acid residues in a 3D-modeling. We report on a 4-week old girl with craniotabes, severe defects of ossification, and failure to thrive. Typical clinical features as low serum alkaline phosphatase, high serum calcium concentration, increased urinary calcium excretion, and nephrocalcinosis were observed. Vitamin D was withdrawn and the patient was started on calcitonin and hydrochlorothiazide. Nonetheless, the girl died at the age of 5 months from respiratory failure. Sequence analysis of the patient's TNSALP gene revealed two heterozygous mutations [c.653T>C (I201T), c.1171C>T (R374C)]. Transfection studies of the unique I201T variant in COS-7 cells yielded a mutant TNSALP protein with only a residual enzyme activity (3.7%) compared with wild-type, whereas the R374C variant was previously shown to reduce normal activity to 10.3%. 3D-modeling of the mutated enzyme showed that I201T resides in a region that does not belong to any known functional site. We note that I201, which has been conserved during evolution, is buried in a hydrophobic pocket and, therefore, the I>T-change should affect its functional properties. Residue R374C is located in the interface between monomers and it has been previously suggested that this mutation affects dimerization. These findings explain the patient's clinical picture and severe course.

Hypophosphatasia

Hypophosphatasia is a rare inherited disorder characterized by defective bone and tooth mineralization, and deficiency of serum and bone alkaline phosphatase activity. The frequency of the disease has been estimated to be one in 100 000 for severe forms, but mild forms of hypophosphatasia may be more common. The symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to early tooth loss without bone symptoms. The transmission of severe forms is autosomal recessive, while milder forms may be transmitted as dominant or recessive autosomal traits. The diagnosis is based on serum alkaline phosphatase assay and molecular analysis of the liver/bone/kidney alkaline phosphatase gene (ALPL). Currently, there is no treatment for the disease. Over the past 10 years, great progress has been made in understanding the structure of tissue non-specific alkaline phosphatase, its function in bone mineralization, and the effect of ALPL mutations responsible for hypophosphatasia.

TEM analysis of apatite surface layers observed on zinc based glass polyalkenoate cements

Glass polyalkenoate cements (GPCs) are acid base cements formed by the reaction of an aqueous solution of polyalkenoic acid, usually polyacrylic acid (PAA) with an acid degradable aluminosilicate glass. The result of the reaction is cement consisting of reacted and unreacted glass particles embedded in a polysalt matrix. In addition to these conventional GPCs, aluminium-free glass polyalkenoate cements based on zinc silicate glasses (Zn-GPCs) exhibit significant potential as bone cements for several reasons. Primarily, they are formulated without the inclusion of aluminium (Al) [1] in the glass phase and thus eliminate clinical complications arising from the release of the Al ion from the cement in vivo. Such complications have, in the past, included aluminium-induced encephalopathy [2– 5] and defective mineralization of cancellous bone [6]. Secondly, Zn-GPCs set without a significant evolution of heat, when compared with commercial bone cements such as Spineplex (Stryker, Limerick, Ireland). Finally, these materials can be tailored to release clinically beneficial ions into the surrounding tissues [7]. In addition to Zn, these cements have been synthesized to contain strontium (Sr) [8, 9]. Both Sr and Zn inhibit osteoclastic turnover and promote osteoblastic turnover, resulting in increased bone strength and decreased fracture risk [10–14]. Conventional GPCs bond directly to hydroxyapatite (HA), the mineral phase of tooth and bone, via the adsorption of carboxylate groups of the polyacid chains into the HA structure [15]. This indicates all conventional GPCs are capable of bonding to the living bone. However, it has been postulated that an essential requirement for a material to bond to living bone lies in its ability to form a bone-like crystalline apatite layer at its surface in vivo and that such a system can be replicated using simulated body fluid (SBF) [16]. Kamitakahara et al. recently used SBF to evaluate the bone bonding ability of conventional GPCs and found that PAA inhibited the formation of an apatite layer at the surface of GPCs and thus concluded that they were unlikely to bond to bone in vivo [17]. Given these conflicting viewpoints, the authors have previously evaluated the ability of Zn-GPCs to form a bone-like apatite layer in vitro using SBF [1, 9]. It was shown that calcium phosphate layers are produced on ZnGPCs immersed in SBF within 24 h. However, thin film Xray diffraction (TF-XRD) indicates no crystallinity within the surface layer, and chemical analysis using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) of the surface layers is complicated by the collection of elemental signatures from the cement sample itself. In order to eliminate these issues, this letter seeks to validate the amorphous nature and composition of the surface layers observed on Zn-GPCs after immersion in SBF using transmission electron microscopy. One glass composition 0.08SrO/0.08CaO/0.36ZnO/ 0.48SiO2 (mol. fraction), was synthesized. Appropriate amounts of analytical grade strontium carbonate, calcium carbonate, zinc oxide and silicon dioxide (Sigma Aldrich, Dublin, Ireland), were weighed out in a plastic tub and mixed in a ball mill for 1 h, then dried (100 C, 1 h). The D. Boyd (&) M. R. Towler A. W. Wren O. M. Clarkin D. A. Tanner Materials and Surface Science Institute, University of Limerick, National Technological Park, Plassey Park, Castletroy, Limerick, Ireland e-mail: Daniel.Boyd@ul.ie

Hypophosphatasia

Hypophosphatasia is a rare inherited disorder characterized by defective bone and teeth mineralization, and deficiency of serum and bone alkaline phosphatase activity. The prevalence of severe forms of the disease has been estimated at 1/100 000.The symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to early loss of teeth without bone symptoms. Depending on the age at diagnosis, six clinical forms are currently recognized: perinatal (lethal), perinatal benign, infantile, childhood, adult and odontohypophosphatasia. In the lethal perinatal form, the patients show markedly impaired mineralization in utero. In the prenatal benign form these symptoms are spontaneously improved. Clinical symptoms of the infantile form are respiratory complications, premature craniosynostosis, widespread demineralization and rachitic changes in the metaphyses. The childhood form is characterized by skeletal deformities, short stature, and waddling gait, and the adult form by stress fractures, thigh pain, chondrocalcinosis and marked osteoarthropathy. Odontohypophosphatasia is characterized by premature exfoliation of fully rooted primary teeth and/or severe dental caries, often not associated with abnormalities of the skeletal system.The disease is due to mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL; OMIM# 171760) encoding the tissue-nonspecific alkaline phosphatase (TNAP). The diagnosis is based on laboratory assays and DNA sequencing of the ALPL gene. Serum alkaline phosphatase (AP) activity is markedly reduced in hypophosphatasia, while urinary phosphoethanolamine (PEA) is increased. By using sequencing, approximately 95% of mutations are detected in severe (perinatal and infantile) hypophosphatasia.Genetic counseling of the disease is complicated by the variable inheritance pattern (autosomal dominant or autosomal recessive), the existence of the uncommon prenatal benign form, and by incomplete penetrance of the trait. Prenatal assessment of severe hypophosphatasia by mutation analysis of chorionic villus DNA is possible. There is no curative treatment for hypophosphatasia, but symptomatic treatments such as non-steroidal anti-inflammatory drugs or teriparatide have been shown to be of benefit. Enzyme replacement therapy will be certainly the most promising challenge of the next few years.

Novel Mouse Model of Autosomal Semidominant Adult Hypophosphatasia Has a Splice Site Mutation in the Tissue Nonspecific Alkaline Phosphatase Gene Akp2

Deactivating mutations in the TNSALP gene cause HPP. Akp2(-/-) mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A-a hypomorphic allele named Akp2(Hpp). The same mutation has been reported in a patient. Akp2(Hpp/+) mice have approximately 50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2(-/-) mice, Akp2(Hpp/Hpp) mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2(Hpp/Hpp) plasma. Osteoblasts display approximately 10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5'-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2(Hpp/Hpp) mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. Akp2(Hpp/Hpp) mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.

Osteoporosis following solid organ transplantation

Damaris Vega & Khashayar Sakhaee† †Author for correspondence University of Texas Southwestern Medical Center, Department of Internal Medicine and, Charles & Jane Pak Center for Mineral Metabolism & Clinical Research, 5323 Harry Hines Boulevard, Dallas, TX 75390-8885, USA Tel.: +1 214 590 7783; Fax: +1 214 590 4091; khashayar.sakhaee@utsouth western.edu ‘Despite the introduction of new immunosuppressive regimens designed for renal allograft recipients ... post-transplantation bone disease remains a major complication in this population.’ Since the introduction of new immunomodulatory agents, the number of organ transplants has increased significantly worldwide. Approximately 28,000 subjects received solid organ transplants in the USA from 2005 to 2006 [101] – approximately double the number of solid organ transplants performed in 1988. Despite the introduction of new immunosuppressive regimens designed for renal allograft recipients, including cyclosporine and tacrolimus, post-transplantation bone disease remains a major complication in this population. It was once thought that the introduction of these new immunomodulatory agents and the limited use of glucocorticoids would ameliorate this complication. However, the sustained loss of bone mineral density and spontaneous bone fractures remain a major cause of morbidity in these subjects [1–5]. The pathophysiologic mechanisms responsible for osteoporosis following organ transplantation are diverse and multifactorial [6]. The most common responsible factors include the underlying conditions leading to transplantation, lifestyle and nutritional changes, hormonal dysregulations, including abnormalities in calcitropic and gonadal hormones, and the skeletal effects of immunomodulatory agents [7–9]. Most commonly, medical immunosuppression has been implicated as the leading catalyst in the development of post-transplantation bone disease [10–13]. However, various renal, hepatic, cardiac and pulmonary diseases also have specific pathophysiologies that may contribute to the outcome of bone disease in this population [14–33]. Careful consideration should be given to the unique clinical picture of each individual patient before and after transplantation. Such a characterization will facilitate optimal management of this diverse group of patients to decrease the incidence of bone fractures and ultimately improve their quality of life. Bone disease is commonly encountered in chronic renal failure patients who experience a continual decline in renal function. Secondary hyperparathyroidism may persist following successful renal transplantation and potentially accentuate the adverse effects of immunosuppressive drugs on the development of bone disease [34]. In addition, the synergistic effect of parathyroid hormone and elevated serum concentrations of phosphatonins, including FGF-23, which has been demonstrated to be elevated after renal transplantation, result in renal phosphorus wasting by reducing renal tubular reabsorption [35–38]. Persistent phosphorus depletion will potentially contribute to defective bone mineralization and progression to osteomalacia and low bone turnover. However, these complications have been largely unnoticed. Therefore, the magnitude of skeletal abnormalities following renal transplantation are much broader than in other solid organ transplantations [39]. These factors may be partly responsible for the continued loss of bone mineral density and increased cumulative risk of new fractures following renal transplantation [3]. Moreover, glucocorticoid treatment, by uncoupling of bone resorption and bone formation, may attenuate the recovery of an impaired bone turnover caused by adynamic bone disease − the most predominant skeletal lesion encountered in patients during dialysis treatment [11,31,40–42].

Prenatal Genetic Diagnosis of Severe Perinatal (Lethal) Hypophosphatasia

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency in tissue-nonspecific alkaline phosphatase (TNSALP) activity. This disorder is caused by various mutations of the TNSALP gene. We report here the prenatal diagnosis of the perinatal (lethal) type of hypophosphatasia in a sibling of an affected infant. The infant had been found to have hypophosphatasia on the basis of both clinical and radiologic manifestations and the finding of a homozygous single T nucleotide deletion at 1559 (1559delT) of the TNSALP gene on molecular analysis. Both parents were carriers with a heterozygous mutation in the same position, although they were not consanguineous. After their next child had been conceived, fetal genomic DNA was extracted from cultured cells of amniotic fluid at 15 weeks' gestation. The fetus had a homozygous 1559delT mutation. An ultrasonography examination at 19 weeks' gestation showed marked hypomineralization of all bony structures. A prenatal genetic diagnosis for hypophosphatasia in combination with ultrasonography is thus considered to be useful for confirming the diagnosis of hypophosphatasia, which presents with a wide variety of phenotypes. As a result, prenatal genetic counseling for hypophosphatasia with collaboration between obstetricians and clinical genetics teams.

Aberrant interchain disulfide bridge of tissue‐nonspecific alkaline phosphatase with an Arg433→Cys substitution associated with severe hypophosphatasia

Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated. Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypophosphatasia, respectively. To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems. In contrast to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALP(R433C). Sucrose density gradient centrifugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type. Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to be present in a free form. Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit. Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C), it strongly inhibited its alkaline phosphatase activity. This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type. Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function. Increased susceptibility to proteases confirmed a gross conformational change of TNSALP(R433C) compared with the wild-type. Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNSALP(R433C).

The Role of Tumor Necrosis Factor α in Down-Regulation of Osteoblast Phex Gene Expression in Experimental Murine Colitis

Reduced bone mass is a common complication of inflammatory bowel disease (IBD), although the mechanisms that contribute to osteopenia are not completely understood. Tumor necrosis factor alpha (TNF-alpha) is up-regulated in patients with IBD and has detrimental effects on osteoblasts. Phex gene is expressed predominantly in osteoblasts, and its disruption results in defective bone mineralization. The aim of this study was to evaluate whether TNF-alpha regulates Phex gene expression thus contributing to the abnormal bone metabolism observed in IBD. Phex gene expression was evaluated in calvaria of 6-7-week-old mice administered with trinitrobenzene sulfonic acid (TNBS) with or without neutralizing anti-TNF-alpha antibody, dietary curcumin, or systemically with recombinant TNF-alpha. TNF-alpha-treated UMR-106 osteoblasts were also examined. Phex promoter activity was assayed in transiently transfected TNF-alpha-treated UMR-106 cells. Compared with control animals, Phex messenger RNA (mRNA) expression decreased by 40%-50% in both TNBS colitis and TNF-alpha-injected mice. Dietary curcumin and anti-TNF-alpha antibody counteracted the detrimental effect of TNBS on Phex gene expression. TNF-alpha-treated UMR-106 cells showed a concentration-dependent and transcriptionally mediated decrease in Phex mRNA and gene promoter activity, with the -133 to -74 bp region of the Phex promoter likely involved in the mechanism of TNF-alpha action. Coinciding with decreased Phex protein level, TNF-alpha drastically reduced mineralization in UMR-106 osteoblasts. Acute colitis and TNF-alpha decrease Phex mRNA and protein expression via a transcriptional mechanism. TNF-alpha-mediated reduction in Phex protein is at least in part responsible for inhibition of osteoblast mineralization, and the described mechanism may contribute to the abnormal bone metabolism associated with IBD.

Osteomalacia

Osteomalacia is characterized by defective mineralization of bone, leading to an accumulation of unmineralized bone matrix (osteoid). Rickets represents the occurrence of this defect in growing children before the closure of epiphysis. In osteomalacia, there is a reduction in the mineralized bone:matrix ratio, unlike osteoporosis, where this ratio remains normal. There is sufficient bone volume, but the bone has reduced calcification. This contribution discusses the: aetiology, clinical features, investigations and management of osteomalacia.

Two Novel PHEX Mutations in Taiwanese Patients with X-Linked Hypophosphatemic Rickets

Backgrounds: X-linked hypophosphatemic rickets (XLH) is an X-linked dominant disease characterized by renal phosphate wasting, hypophosphatemia, aberrant vitamin D metabolism, and defective bone mineralization. The disease is caused by mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) located at Xp22.1. To date, a variety of PHEX mutations have been identified in these patients. Methods: PCR and direct sequencing was performed for all exons and intron-exon boundaries of the PHEX gene in two XLH families. Results: Two novel mutations, including a missense mutation (L206W) in exon 5 and a frameshift mutation (nucleotide 1826_1830delAAAAG, stop after codon 610) in exon 18 were discovered and the laboratory and radiographic findings for these patients analyzed. Conclusions: We found that PHEX gene mutations were responsible for XLH in these Taiwanese patients. Additional studies are needed to enhance understanding of the role of PHEX in XLH pathogenesis.

Characterization of Missense Mutations and Large Deletions in theALPLGene by Sequencing and Quantitative Multiplex PCR of Short Fragments

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.

Downregulation of osteoblast Phex expression by PTH

Human/murine X-linked hypophosphatemia is a dominant disorder associated with renal phosphate wasting and defective bone mineralization. This disorder results from mutations in the PHEX/ Phex ( Phosphate-regulating gene with homologies to endopeptidases on the X chromosome) gene, which is expressed in fully differentiated osteoblasts. The purpose of the present study was to assess whether PTH, a major regulator of bone development and turnover, modulates osteoblastic Phex expression. The effects of different concentrations of PTH (rat fragment 1–34) were determined on Phex mRNA and protein expression in vitro using MC3T3-E1 osteoblastic cells and mouse primary osteoblasts; and in vivo using 45-day-old mice infused for 3 days with PTH. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relative to GAPDH mRNA levels. Phex protein levels were analyzed by immunoprecipitation of 35S-methionine-labeled osteoblast lysates or by immunoblotting of calvaria membrane extracts using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide. Fully differentiated MC3T3-E1 cells were incubated for 4 to 48 h with increasing concentrations of PTH (10 −11 to 10 −7 M). PTH inhibited Phex mRNA expression in both mineralizing and nonmineralizing osteoblast cultures in a dose- and time-dependent manner with a maximal inhibition at 10 −7 M PTH after 24 h (15 ± 7% of control levels, n = 5, P < 0.001). The PTH-mediated downregulation of Phex mRNA levels was associated with corresponding decreases in Phex protein synthesis and suppression at 10 −7 M PTH. Similar results were obtained with primary osteoblasts isolated from newborn mouse calvaria. Consistent with the in vitro findings, continuous PTH infusion to mice elicited decreases in Phex expression in calvaria. The effect of PTH was also assessed on matrix mineralization by mature MC3T3-E1 cells by measuring 45Ca accumulation in cell layers. PTH (10 −7 M) inhibited the initiation (57 ± 2% of control levels, n = 5, P < 0.001) and the progression of matrix mineralization (75 ± 1% of control levels, n = 5, P < 0.001). In summary, PTH inhibits osteoblastic Phex expression in vitro and in vivo. The downregulation of Phex expression by PTH in vitro is associated with inhibition of matrix mineralization, consistent with a role for Phex in bone mineralization.

146 Perinatal Hypophosphatasia - Prenatal Diagnosis

Background: Hypophosphatasia is a rare autosomal recessive disorder characterized by defective bone and teeth mineralization, and deficiency of bone and serum alkaline phosphatase activity (ALP). The clinical expression is highly variable and ranges from stillbirth without mineralized bone to pathologic fractures developing only in late childhood. The responsible gene encoding the tissue non-specific alkaline phosphatase (TNSALP) consists of 12 exons and is localized on chromosome 1p36.1. More than 138 distinct mutations including a great number of compound heterozygous genotypes have been described. Case report: The patient was the first child of unrelated healthy Turkish parents. At 30 weeks of gestation ultrasound examination showed polyhydramnion, short limbs, reduced mineralization of the skull, and short ribs with a hypoplastic chest. Hypophosphatasia was suspected. Analysis of fetal cord blood showed absence of ALP-activity. These findings prompted us to initiate molecular analysis of the TSNALP-gene which revealed compound heterozygous mutations in the TNSALP-gene (G203V and R206Q) (E. Mornet, Versailles, France). The girl was born at 36 weeks gestational age and died of respiratory failure 30 minutes after birth. Clinically, she presented with a large head, soft calotte, muscular hypotonia, short and hyperflexible limbs. X-ray examination proved a severely reduced mineralisation of the bones with marked shortening of the long bones, very short and thin ribs and a hypoplastic chest and lungs. Conclusion: Reduced mineralization especially of the skull and the absence of ALP led to the clinical diagnosis of hypophosphatasia. The mutations found in the fetus predicted a lethal outcome. Prenatal molecular diagnosis is important for the perinatal management.