An Unusual Combination of Neurological Manifestations and Sudden Vision Loss in a Child with Familial Hyperphosphatemic Tumoral Calcinosis

Hyperphosphatemia in the absence of renal failure is an unusual occurrence, particularly in children, but is a common primary feature of familial hyperphosphatemic tumor calcinosis. We report a child with hyperphosphatemia who presented with multiple episodes of neurologic dysfunction involving lower motor neuron facial nerve palsy along with sequential visual loss. He also had an episode of stroke. There was an extensive metastatic calcification of soft tissue and vasculature. Hyperphosphatemia with normal serum alkaline phosphatase, calcium, parathyroid hormone, and renal function was noted. He was managed with hemodialysis and sevelamer (3 months) without much success in reducing serum phosphate level, requiring continuous ambulatory peritoneal dialysis (3 years). Intact fibroblast growth factor 23 (FGF23) was undetectable, with C-terminal FGF23 fragments significantly elevated (2575 RU/ml, normal A (p.N162K) mutation in FGF23 exon 3, confirming the diagnoses of primary FGF23 deficiency, the first case to be reported from India

Serum fibroblast growth factor 23, serum iron and bone mineral density in premenopausal women

Fibroblast growth factor 23 (FGF23) circulates as active protein and inactive fragments. Low iron status increases FGF23 gene expression, and iron deficiency is common. We hypothesized that in healthy premenopausal women, serum iron influences C-terminal and intact FGF23 concentrations, and that iron and FGF23 associate with bone mineral density (BMD). Serum iron, iron binding capacity, percent iron saturation, phosphorus, and other biochemistries were measured in stored fasting samples from healthy premenopausal white (n=1898) and black women (n=994), age 20-55years. Serum C-terminal and intact FGF23 were measured in a subset (1631 white and 296 black women). BMD was measured at the lumbar spine and femur neck. Serum phosphorus, calcium, alkaline phosphatase and creatinine were lower in white women than black women (p<0.001). Serum iron (p<0.0001) and intact FGF23 (p<0.01) were higher in white women. C-terminal FGF23 did not differ between races. Phosphorus correlated with intact FGF23 (white women, r=0.120, p<0.0001; black women r=0.163, p<0.01). However, phosphorus correlated with C-terminal FGF23 only in black women (r=0.157, p<0.01). Intact FGF23 did not correlate with iron. C-terminal FGF23 correlated inversely with iron (white women r=-0.134, p<0.0001; black women r=-0.188, p<0.01), having a steeper slope at iron <50mcg/dl than ≥50mcg/dl. Longitudinal changes in iron predicted changes in C-terminal FGF23. Spine BMD correlated with iron negatively (r=-0.076, p<0.01) in white women; femur neck BMD correlated with iron negatively (r=-0.119, p<0.0001) in black women. Both relationships were eliminated in weight-adjusted models. BMD did not correlate with FGF23. Serum iron did not relate to intact FGF23, but was inversely related to C-terminal FGF23. Intact FGF23 correlated with serum phosphorus. In weight-adjusted models, BMD was not related to intact FGF23, C-terminal FGF23 or iron. The influence of iron on FGF23 gene expression is not important in determining bone density in healthy premenopausal women

Interferon Gamma, but not Calcitriol Improves the Osteopetrotic Phenotypes in ADO2 Mice

ADO2 is a heritable osteosclerotic disorder that usually results from heterozygous missense dominant negative mutations in the chloride channel 7 gene (CLCN7). ADO2 is characterized by a wide range of features and severity, including multiple fractures, impaired vision due to secondary bony overgrowth and/or the lack of the optical canal enlargement with growth, and osteonecrosis/osteomyelitis. The disease is presently incurable, although anecdotal evidence suggests that calcitriol and interferon gamma-1b (IFN-G) may have some beneficial effects. To identify the role of these drugs for the treatment of ADO2, we utilized a knock-in (G213R mutation in Clcn7) ADO2 mouse model that resembles the human disease. Six-week-old ADO2 heterozygous mice were administered vehicle (PBS) or calcitriol or IFN-G 5 times per week for 8 weeks. We determined bone phenotypes using DXA and μCT, and analyzed serum biochemistry and bone resorption markers. ADO2 mice treated with all doses of IFN-G significantly (p<0.05) attenuated the increase of whole body aBMD and distal femur BV/TV gain in both male and female compared to the vehicle group. In contrast, mice treated with low and medium doses of calcitriol showed a trend of higher aBMD and BV/TV whereas high dose calcitriol significantly (p<0.05) increased bone mass compared to the vehicle group. The calcium and phosphorus levels did not differ between vehicle and IFN-G or calcitriol treated mice; however, we detected significantly (p<0.05) elevated levels of CTX/TRAP5b ratio in IFN-G treated mice. Our findings indicate that while IFN-G at all doses substantially improved the osteopetrotic phenotypes in ADO2 heterozygous mice, calcitriol treatment at any dose did not improve the phenotype and at high dose further increased bone mass. Thus, use of high dose calcitriol therapy in ADO2 patients merits serious reconsideration. Importantly, our data support the prospect of a clinical trial of IFN-G in ADO2 patients

Genome-wide association study of serum iron phenotypes in premenopausal women of European descent

A genome-wide association study was performed in 1,130 premenopausal women to detect common variants associated with three serum iron-related phenotypes. Total iron binding capacity was strongly associated (p=10−14) with variants in and near the TF gene (transferrin), the serum iron transporting protein, and with variants in HFE (p= 4×10−7), which encodes the human hemochromatosis gene. Association was also detected between percent iron saturation (p=10−8) and variants in the chromosome 6 region containing both HFE and SLC17A2, which encodes a phosphate transport protein. No significant associations were detected with serum iron, but variants in HFE were suggestive (p=10−6). Our results corroborate prior studies in older subjects and demonstrate that the association of these genetic variants with iron phenotypes can be detected in premenopausal women

Oral Iron Replacement Normalizes Fibroblast Growth Factor 23 in Iron Deficient Patients with Autosomal Dominant Hypophosphatemic Rickets

Autosomal dominant hypophosphatemic rickets (ADHR) is caused by mutations impairing cleavage of fibroblast growth factor 23 (FGF23). FGF23 gene expression increases during iron deficiency. In humans and mice with the ADHR mutation, iron deficiency results in increased intact FGF23 concentrations and hypophosphatemia. We conducted a prospective open label pilot clinical trial of oral iron replacement over 12 months in ADHR patients to test the hypothesis that oral iron administration would normalize FGF23 concentrations. Eligibility criteria included: FGF23 mutation; and either serum iron 30 pg/mL at screening. Key exclusion criteria were kidney disease and pregnancy. Oral iron supplementation started at 65 mg daily and was titrated based on fasting serum iron concentration. The primary outcome was decrease in fasting intact FGF23 by ≥20% from baseline. Six adults (three male, three female) having the FGF23-R176Q mutation were enrolled; five completed the 12-month protocol. At baseline three of five subjects had severely symptomatic hypophosphatemia (phosphorus <2.5 mg/dL) and received calcitriol with or without phosphate concurrent with oral iron during the trial. The primary outcome was met by 4 of 5 (80%) subjects all by month 4, and 5 of 5 had normal intact FGF23 at month 12. Median (minimum, maximum) intact FGF23 concentration decreased from 172 (20, 192) pg/mL at baseline to 47 (17, 78) pg/mL at month 4 and 42 (19, 63) pg/mL at month 12. Median ferritin increased from 18.6 (7.7, 82.5) ng/mL at baseline to 78.0 (49.6, 261.0) ng/mL at month 12. During iron treatment, all three subjects with baseline hypophosphatemia normalized serum phosphorus, had markedly improved symptoms, and were able to discontinue calcitriol and phosphate. Oral iron repletion normalized FGF23 and phosphorus in symptomatic, iron-deficient ADHR subjects. Thus, the standard approach to ADHR should include recognition, treatment, and prevention of iron deficiency

Interferon Gamma-1b Does Not Increase Markers of Bone Resorption in Autosomal Dominant Osteopetrosis

In autosomal dominant osteopetrosis type 2 (ADO2) CLCN7 mutations cause impaired osteoclast function. Severe consequences include skeletal fragility despite high bone mass, osteomyelitis, osteonecrosis, bone marrow failure, and severe cranial nerve impingement. There is no effective medical treatment for ADO2. We recruited subjects with ADO2 into a 14-week, open-label, pilot clinical trial of interferon gamma-1b. Doses were titrated based on tolerability and if fasting serum C-telopeptide (CTX) was <25% above baseline at week 8, targeting doses of 100 mcg/m2 three times a week. The primary outcomes were change from baseline in CTX and N-telopeptide/creatinine ratio (NTX/Cr) at week 14. Secondary outcomes included changes in urine calcium/creatinine ratio, bone formation markers and tolerability. Nine adults and 3 children were recruited. Severe manifestations of ADO2 included histories of fractures (100%), osteomyelitis (16.7%), vision loss (50%), and anemia (58.3%). Baseline CTX and NTX/Cr were generally low-normal. Procollagen type I N-terminal propeptide was elevated or in the upper-normal range in 11/12 (91.6%) subjects. Elevations of AST and LDH were common. One subject withdrew due to rash. Five subjects achieved doses of 50 ug/m2 three days a week, while 6 reached the full dose of 100 ug/m2 three days a week. Only 3/11 (27.3%) completing subjects achieved the primary outcome of increasing CTX ≥25% above baseline at week 14. The mean change from baseline in CTX at week 14 was +2.2% (SD 43.2%, p=0.86). Likewise, there was no significant change in NTX/Cr (mean change −2.1%, p=0.81). Interferon gamma-1b was poorly tolerated. Most subjects had adverse events, and the Mental Health and Mental Component Scales of the SF-36v2 declined slightly (p<0.05). Over 14 weeks, interferon gamma-1b failed to significantly increase bone turnover markers in ADO2 and was poorly tolerated. Consequently, interferon gamma-1b is unlikely to be effective for decreasing bone mass in ADO2

Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes

Recently, we demonstrated that osteoblast-specific overexpression of human WNT16 increased both cortical and trabecular bone mass and structure in mice. To further identify the cell-specific role of Wnt16 in bone homeostasis, we created transgenic (TG) mice overexpressing human WNT16 in osteocytes using Dmp1 promoter (Dmp1-hWNT16 TG) on C57BL/6 (B6) background. We analyzed bone phenotypes and serum bone biomarkers, performed gene expression analysis and measured dynamic bone histomorphometry in Dmp1-hWNT16 TG and wild-type (WT) mice. Compared to WT mice, Dmp1-hWNT16 TG mice exhibited significantly higher whole-body, spine and femoral aBMD, BMC and trabecular (BV/TV, Tb.N, and Tb.Th) and cortical (bone area and thickness) parameters in both male and female at 12 weeks of age. Femur stiffness and ultimate force were also significantly improved in the Dmp1-hWNT16 TG female mice, compared to sex-matched WT littermates. In addition, female Dmp1-hWNT16 TG mice displayed significantly higher MS/BS, MAR and BFR/BS compared to the WT mice. Gene expression analysis demonstrated significantly higher mRNA level of Alp in both male and female Dmp1-hWNT16 TG mice and significantly higher levels of Osteocalcin, Opg and Rankl in the male Dmp1-hWNT16 TG mice in bone tissue compared to sex-matched WT mice. These results indicate that WNT16 plays a critical role for acquisition of both cortical and trabecular bone mass and strength. Strategies designed to use WNT16 as a target for therapeutic interventions will be valuable to treat osteoporosis and other low bone mass conditions

Serum fibroblast growth factor 23, serum iron and bone mineral density in premenopausal women

Fibroblast growth factor 23 (FGF23) circulates as active protein and inactive fragments. Low iron status increases FGF23 gene expression, and iron deficiency is common. We hypothesized that in healthy premenopausal women, serum iron influences C-terminal and intact FGF23 concentrations, and that iron and FGF23 associate with bone mineral density (BMD). Serum iron, iron binding capacity, percent iron saturation, phosphorus, and other biochemistries were measured in stored fasting samples from healthy premenopausal white (n=1898) and black women (n=994), age 20-55years. Serum C-terminal and intact FGF23 were measured in a subset (1631 white and 296 black women). BMD was measured at the lumbar spine and femur neck. Serum phosphorus, calcium, alkaline phosphatase and creatinine were lower in white women than black women (p<0.001). Serum iron (p<0.0001) and intact FGF23 (p<0.01) were higher in white women. C-terminal FGF23 did not differ between races. Phosphorus correlated with intact FGF23 (white women, r=0.120, p<0.0001; black women r=0.163, p<0.01). However, phosphorus correlated with C-terminal FGF23 only in black women (r=0.157, p<0.01). Intact FGF23 did not correlate with iron. C-terminal FGF23 correlated inversely with iron (white women r=-0.134, p<0.0001; black women r=-0.188, p<0.01), having a steeper slope at iron <50mcg/dl than ≥50mcg/dl. Longitudinal changes in iron predicted changes in C-terminal FGF23. Spine BMD correlated with iron negatively (r=-0.076, p<0.01) in white women; femur neck BMD correlated with iron negatively (r=-0.119, p<0.0001) in black women. Both relationships were eliminated in weight-adjusted models. BMD did not correlate with FGF23. Serum iron did not relate to intact FGF23, but was inversely related to C-terminal FGF23. Intact FGF23 correlated with serum phosphorus. In weight-adjusted models, BMD was not related to intact FGF23, C-terminal FGF23 or iron. The influence of iron on FGF23 gene expression is not important in determining bone density in healthy premenopausal women

Oral Iron Replacement Normalizes Fibroblast Growth Factor 23 in Iron Deficient Patients with Autosomal Dominant Hypophosphatemic Rickets

Autosomal dominant hypophosphatemic rickets (ADHR) is caused by mutations impairing cleavage of fibroblast growth factor 23 (FGF23). FGF23 gene expression increases during iron deficiency. In humans and mice with the ADHR mutation, iron deficiency results in increased intact FGF23 concentrations and hypophosphatemia. We conducted a prospective open label pilot clinical trial of oral iron replacement over 12 months in ADHR patients to test the hypothesis that oral iron administration would normalize FGF23 concentrations. Eligibility criteria included: FGF23 mutation; and either serum iron 30 pg/mL at screening. Key exclusion criteria were kidney disease and pregnancy. Oral iron supplementation started at 65 mg daily and was titrated based on fasting serum iron concentration. The primary outcome was decrease in fasting intact FGF23 by ≥20% from baseline. Six adults (three male, three female) having the FGF23-R176Q mutation were enrolled; five completed the 12-month protocol. At baseline three of five subjects had severely symptomatic hypophosphatemia (phosphorus <2.5 mg/dL) and received calcitriol with or without phosphate concurrent with oral iron during the trial. The primary outcome was met by 4 of 5 (80%) subjects all by month 4, and 5 of 5 had normal intact FGF23 at month 12. Median (minimum, maximum) intact FGF23 concentration decreased from 172 (20, 192) pg/mL at baseline to 47 (17, 78) pg/mL at month 4 and 42 (19, 63) pg/mL at month 12. Median ferritin increased from 18.6 (7.7, 82.5) ng/mL at baseline to 78.0 (49.6, 261.0) ng/mL at month 12. During iron treatment, all three subjects with baseline hypophosphatemia normalized serum phosphorus, had markedly improved symptoms, and were able to discontinue calcitriol and phosphate. Oral iron repletion normalized FGF23 and phosphorus in symptomatic, iron-deficient ADHR subjects. Thus, the standard approach to ADHR should include recognition, treatment, and prevention of iron deficiency

The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients