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Item Autoimmune hyperphosphatemic tumoral calcinosis in a patient with FGF23 autoantibodies(American Society for Clinical Investigation, 2018-12-03) Roberts, Mary Scott; Burbelo, Peter D.; Egli-Spichtig, Daniela; Perwad, Farzana; Romero, Christopher J.; Ichikawa, Shoji; Farrow, Emily; Econs, Michael J.; Guthrie, Lori C.; Collins, Michael T.; Gafni, Rachel I.; Medicine, School of MedicineHyperphosphatemic familial tumoral calcinosis (HFTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is an autosomal recessive disorder of ectopic calcification due to deficiency of or resistance to intact fibroblast growth factor 23 (iFGF23). Inactivating mutations in FGF23, N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO (KL) have been reported as causing HFTC/HHS. We present what we believe is the first identified case of autoimmune hyperphosphatemic tumoral calcinosis in an 8-year-old boy. In addition to the classical clinical and biochemical features of hyperphosphatemic tumoral calcinosis, the patient exhibited markedly elevated intact and C-terminal FGF23 levels, suggestive of FGF23 resistance. However, no mutations in FGF23, KL, or FGF receptor 1 (FGFR1) were identified. He subsequently developed type 1 diabetes mellitus, which raised the possibility of an autoimmune cause for hyperphosphatemic tumoral calcinosis. Luciferase immunoprecipitation systems revealed markedly elevated FGF23 autoantibodies without detectable FGFR1 or Klotho autoantibodies. Using an in vitro FGF23 functional assay, we found that the FGF23 autoantibodies in the patient's plasma blocked downstream signaling via the MAPK/ERK signaling pathway in a dose-dependent manner. Thus, this report describes the first case, to our knowledge, of autoimmune hyperphosphatemic tumoral calcinosis with pathogenic autoantibodies targeting FGF23. Identification of this pathophysiology extends the etiologic spectrum of hyperphosphatemic tumoral calcinosis and suggests that immunomodulatory therapy may be an effective treatment.Item Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis(2011-12) Ichikawa, Shoji; Austin, Anthony M.; Gray, Amie K.; Allen, Matthew R.; Econs, Michael J.Mutations in the GALNT3 gene cause tumoral calcinosis characterized by ectopic calcifications due to persistent hyperphosphatemia. We recently developed Galnt3 knockout mice in a mixed background, which had hyperphosphatemia with increased bone mineral density (BMD) and infertility in males. To test the effect of dietary phosphate intake on their phenotype, Galnt3 knockout mice were generated in the C57BL/6J strain and fed various phosphate diets: 0.1% (low), 0.3% (low normal), 0.6% (normal), and 1.65% (high). Sera were analyzed for calcium, phosphorus, alkaline phosphatase, creatinine, blood urine nitrogen, 1,25-dihydroxyvitamin D, osteocalcin, tartrate-resistant acid phosphatase 5b, and fibroblast growth factor 23 (Fgf23). Femurs were evaluated by dual-energy x-ray absorptiometry, dynamic histomorphometry, and/or microcomputed tomography. Galnt3 knockout mice in C57BL/6J had the same biochemical phenotype observed in our previous study: hyperphosphatemia, inappropriately normal 1,25-dihydroxyvitamin D level, decreased alkaline phosphatase activity, and low intact Fgf23 concentration but high Fgf23 fragments. Skeletal analyses of their femurs revealed significantly high BMD with increased cortical bone area and trabecular bone volume. On all four phosphate diets, Galnt3 knockout mice had consistently higher phosphorus levels and lower alkaline phosphatase and intact Fgf23 concentrations than littermate controls. The low-phosphate diet normalized serum phosphorus, alkaline phosphatase, and areal BMD but failed to correct male infertility in Galnt3 knockout mice. The high-phosphate diet did not increase serum phosphorus concentration in either mutant or control mice due to a compensatory increase in circulating intact Fgf23 levels. In conclusion, dietary phosphate restriction normalizes biochemical and skeletal phenotypes of Galnt3 knockout mice and, thus, can be an effective therapy for tumoral calcinosis.Item Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects(2010-07) Yamazaki, Yuji; Imura, Akihiro; Urakawa, Itaru; Shimada, Takashi; Murakami, Junko; Aono, Yukiko; Hasegawa, Hisashi; Yamashita, Takeyoshi; Nakatani, Kimihiko; Saito, Yoshihiko; Okamoto, Nozomi; Kurumatani, Norio; Namba, Noriyuki; Kitaoka, Taichi; Ozono, Keiichi; Sakai, Tomoyuki; Hataya, Hiroshi; Ichikawa, Shoji; Imel, Erik A.; Econs, Michael J.; Nabeshima, Yo-ichiBackground α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca2+) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation. Objectives The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and (2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. Results We successively developed an ELISA to measure serum sαKl in healthy volunteers (n = 142, males 66) of ages (61.1 ± 18.5 year). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)2D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL). Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n = 39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age-matched controls. Conclusion We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism.Item FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells(Oncotarget, 2015-08-14) Suvannasankha, Attaya; Tompkins, Douglas R.; Edwards, Daniel F.; Petyaykina, Katarina V.; Crean, Colin D.; Fournier, Pierrick G.; Parker, Jamie M.; Sandusky, George E.; Ichikawa, Shoji; Imel, Erik A.; Chirgwin, John M.; Department of Medicine, IU School of MedicineMultiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.Item Generation of the first Autosomal Dominant Osteopetrosis Type II (ADO2) disease models(Elsevier B.V., 2014-02) Alam, Imranul; Gray, Amie K.; Chu, Kang; Ichikawa, Shoji; Mohammad, Khalid S.; Capannolo, Marta; Capulli, Mattia; Maurizi, Antonio; Muraca, Maurizio; Teti, Anna; Econs, Michael J.; Del Fattore, Andrea; Department of Orthopaedic Surgery, IU School of MedicineAutosomal Dominant Osteopetrosis Type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl−/1H+ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.Item Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse(Endocrine Society, 2014-10) Ichikawa, Shoji; Gray, Amie K.; Padgett, Leah R.; Allen, Matthew R.; Clinkenbeard, Erica L.; Sarpa, Nicole M.; White, Kenneth E.; Econs, Michael J.; Department of Medicine, IU School of MedicineFibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in Galnt3 knockout mice.Item Intronic deletions in the SLC34A3 gene: A cautionary tale for mutation analysis of hereditary hypophosphatemic rickets with hypercalciuria(Elsevier B.V., 2014-02) Ichikawa, Shoji; Tuchman, Shamir; Padgett, Leah R.; Gray, Amie K.; Baluarte, H. Jorge; Econs, Michael J.; Department of Medicine, IU School of MedicineHereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder, characterized by hypophosphatemia, variable degrees of rickets/osteomalacia, and hypercalciuria secondary to increased serum 1,25-dihydroxyvitamin D [1,25(OH)2D] levels. HHRH is caused by mutations in the SLC34A3 gene, which encodes sodium-phosphate co-transporter type IIc. A 6 ½-year-old female presented with a history of nephrolithiasis. Her metabolic evaluation revealed increased 24- hour urine calcium excretion with high serum calcium, low intact parathyroid hormone (PTH) levels, and elevated 1,25(OH)2D level. In addition, the patient had low to low-normal serum phosphorus with high urine phosphorus. The patient had normal stature; without rachitic or boney deformities or a history of fractures. Genetic analysis of SLC34A3 revealed the patient to be a compound heterozygote for a novel single base pair deletion in exon 12 (c.1304delG) and 30-base pair deletion in intron 6 (g.1440–1469del). The single-base pair mutation causes a frameshift, which results in premature stop codon. The intronic deletion is likely caused by misalignment of the 4-basepair homologous repeats and results in the truncation of an already small intron to 63 bp, which would impair proper RNA splicing of the intron. This is the fourth unique intronic deletion identified in patients with HHRH, suggesting the frequent occurrence of sequence misalignments in SLC34A3 and the importance of screening introns in patients with HHRH.Item Mutations in SLC34A3/NPT2c are associated with kidney stones and nephrocalcinosis(American Society of Nephrology, 2014-10) Dasgupta, Debayan; Wee, Mark J.; Reyes, Monica; Li, Yuwen; Simm, Peter J.; Sharma, Amita; Schlingmann, Karl-Peter; Janer, Marco; Biggin, Andrew; Lazier, Joanna; Gessner, Michaela; Chrysis, Dionisios; Tuchman, Shamir; Baluarte, H. Jorge; Levine, Michael A.; Tiosano, Dov; Insogna, Karl; Hanley, David A.; Carpenter, Thomas O.; Ichikawa, Shoji; Hoppe, Bernd; Konrad, Martin; Sävendahl, Lars; Munns, Craig F.; Lee, Hang; Jüppner, Harald; Bergwitz, Clemens; Department of Medicine, IU School of MedicineCompound heterozygous and homozygous (comp/hom) mutations in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium (Na(+))-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate wasting resulting in hypophosphatemia, correspondingly elevated 1,25(OH)2 vitamin D levels, hypercalciuria, and rickets/osteomalacia. Similar, albeit less severe, biochemical changes are observed in heterozygous (het) carriers and indistinguishable from those changes encountered in idiopathic hypercalciuria (IH). Here, we report a review of clinical and laboratory records of 133 individuals from 27 kindreds, including 5 previously unreported HHRH kindreds and two cases with IH, in which known and novel SLC34A3 mutations (c.1357delTTC [p.F453del]; c.G1369A [p.G457S]; c.367delC) were identified. Individuals with mutations affecting both SLC34A3 alleles had a significantly increased risk of kidney stone formation or medullary nephrocalcinosis, namely 46% compared with 6% observed in healthy family members carrying only the wild-type SLC34A3 allele (P=0.005) or 5.64% in the general population (P<0.001). Renal calcifications were also more frequent in het carriers (16%; P=0.003 compared with the general population) and were more likely to occur in comp/hom and het individuals with decreased serum phosphate (odds ratio [OR], 0.75, 95% confidence interval [95% CI], 0.59 to 0.96; P=0.02), decreased tubular reabsorption of phosphate (OR, 0.41; 95% CI, 0.23 to 0.72; P=0.002), and increased serum 1,25(OH)2 vitamin D (OR, 1.22; 95% CI, 1.05 to 1.41; P=0.008). Additional studies are needed to determine whether these biochemical parameters are independent of genotype and can guide therapy to prevent nephrocalcinosis, nephrolithiasis, and potentially, CKD.Item Nicotinamide treatment in a murine model of familial tumoral calcinosis reduces serum Fgf23 and raises heart calcium(Elsevier, 2014-10) Reilly, Austin M.; Gray, Amie K.; Moe, Sharon M.; Ichikawa, Shoji; Department of Medicine, IU School of MedicineMutations in the GALNT3 gene result in familial tumoral calcinosis, characterized by persistent hyperphosphatemia and ectopic calcific masses in soft tissues. Since calcific masses often recur after surgical removal, a more permanent solution to the problem is required. Nicotinamide is reported to lower serum phosphate by decreasing sodium-dependent phosphate co-transporters in the gut and kidney. However, its effectiveness in tumoral calcinosis remains unknown. In this study, we investigated nicotinamide as a potential therapy for tumoral calcinosis, using a murine model of the disease-Galnt3 knockout mice. Initially, five different doses of nicotinamide were given to normal heterozygous mice intraperitoneally or orally. Treatment had no effect on serum phosphate levels, but serum levels of a phosphaturic hormone, fibroblast growth factor 23 (Fgf23), decreased in a dose-dependent manner. Subsequently, high-dose nicotinamide (40mM) was tested in Galnt3 knockout mice fed a high phosphate diet. The radiographic data pre- and post-treatment showed that nicotinamide did not reverse the calcification. However, the treatment retarded calcification growth after 4weeks, while in the untreated animals, calcifications increased in size. The therapy did not affect serum phosphate levels, but intact Fgf23 decreased in the treated mice. The treated mice also had increased calcium in the heart. In summary, nicotinamide did not alter serum phosphate levels, likely due to compensatory decrease in Fgf23 to counteract the phosphate lowering effect of nicotinamide. Although increased calcium accumulation in the heart is a concern, the therapy appears to slow down the progression of ectopic calcifications.Item Phenotypic and Genotypic Characterization and Treatment of a Cohort with Familial Tumoral Calcinosis/Hyperostosis-Hyperphosphatemia Syndrome(Wiley, 2016-10) Ramnitz, Mary Scott; Gourh, Pravitt; Goldbach-Mansky, Raphaela; Wodajo, Felasfa; Ichikawa, Shoji; Econs, Michael J.; White, Kenneth; Molinolo, Alfredo; Chen, Marcus Y.; Heller, Theo; Del Rivero, Jaydira; Seo-Mayer, Patricia; Arabshahi, Bita; Jackson, Malaka B.; Hatab, Sarah; McCarthy, Edward; Guthrie, Lori C.; Brillante, Beth A.; Gafni, Rachel I.; Collins, Michael T.; Medicine, School of MedicineFamilial tumoral calcinosis (FTC)/hyperostosis-hyperphosphatemia syndrome (HHS) is a rare disorder caused by mutations in the genes encoding fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or KLOTHO. The result is functional deficiency of, or resistance to, intact FGF23 (iFGF23), causing hyperphosphatemia, increased renal tubular reabsorption of phosphorus (TRP), elevated or inappropriately normal 1,25-dihydroxyvitamin D3 (1,25D), ectopic calcifications and/or diaphyseal hyperostosis. Eight subjects with FTC/HHS were studied and treated. Clinical manifestations varied, even within families, ranging from asymptomatic to large, disabling calcifications. All subjects had hyperphosphatemia, increased TRP, and elevated or inappropriately normal 1,25D. C-terminal FGF23 was markedly elevated while iFGF23 was comparatively low, consistent with increased FGF23 cleavage. Radiographs ranged from diaphyseal hyperostosis to massive calcification. Two subjects with severe calcifications also had overwhelming systemic inflammation and elevated C-reactive protein (CRP). GALNT3 mutations were identified in 7 subjects; no causative mutation was found in the eighth. Biopsies from 4 subjects showed ectopic calcification and chronic inflammation, with areas of heterotopic ossification observed in 1 subject. Treatment with low phosphate diet, phosphate binders, and phosphaturia-inducing therapies was prescribed with variable response. One subject experienced complete resolution of a calcific mass after 13 months of medical treatment. In the 2 subjects with systemic inflammation, interleukin-1 (IL-1) antagonists significantly decreased CRP levels with resolution of calcinosis cutis and peri-lesional inflammation in one subject and improvement of overall well-being in both subjects. This cohort expands the phenotype and genotype of FTC/HHS and demonstrates the range of clinical manifestations despite similar biochemical profiles and genetic mutations. Overwhelming systemic inflammation has not been described previously in FTC/HHS; the response to IL-1 antagonists suggests that anti-inflammatory drugs may be useful adjuvants. In addition, this is the first description of heterotopic ossification reported in FTC/HHS, possibly mediated by the adjacent inflammation.