A G‐protein Subunit‐α11 Loss‐of‐Function Mutation,Thr54Met,Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα₁₁) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα₁₁, encoded by GNA11, and to date only two FHH2‐associated Gα₁₁ missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα₁₁ variant was assessed by homology modeling of the related Gα_q protein and by measuring the CaSR‐mediated intracellular calcium (Ca²⁺_i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca²⁺_o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα₁₁ helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα₁₁ in HEK293 cells stably expressing CaSR demonstrate that the Ca²⁺_i responses after stimulation with Ca²⁺_o of the mutant Met54 Gα₁₁ led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC₅₀) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC₅₀ of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα₁₁ mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα₁₁ interdomain interface in CaSR signaling and Ca²⁺_o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Identification of a G‐Protein Subunit‐α11 Gain‐of‐Function Mutation,Val340Met,in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2)

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα₁₁). To date Gα₁₁ mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα₁₁ mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα₁₁ proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα₁₁ led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC₅₀) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC₅₀ of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα₁₁ mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα₁₁ C‐terminal region for G‐protein function and CaSR signal transduction. © 2016 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Familial Hypocalciuric Hypercalcemia as an Atypical Form of Primary Hyperparathyroidism

Familial hypocalciuric hypercalcemia (FHH) causes lifelong hypercalcemia with features that overlap with typical primary hyperparathyroidism (PHPT). The incompleteness of this overlap has led to divergent nomenclatures for FHH. I compare two nomenclatures. One sets FHH as an entity distinct from PHPT. The other groups FHH with PHPT but conditions FHH as atypical PHPT. I analyzed selected articles about calcium‐sensing receptors, FHH, PHPT, CASR, GNA11, and AP2S1. FHH usually results from a heterozygous germline inactivating mutation of the CASR, and less frequently from mutation of GNA11 or AP2S1. The CASR encodes the calcium‐sensing receptors. These are highly expressed on parathyroid cells, where they sense serum calcium concentration and regulate suppression of PTH secretion by serum calcium. Their mutated expression in the kidney in FHH causes increased renal tubular reabsorption of calcium (hypocalciuria). Many FHH features are shared with PHPT and thus support FHH as a form of PHPT. These include a driver mutation expressed mainly in the parathyroid cells. The mutation causes a parathyroid cell insensitivity to extracellular calcium in vivo and in vitro, a right‐shift of the set point for suppression of PTH secretion by calcium. Serum PTH is normal or mildly elevated; ie, it is not appropriately suppressed by hypercalcemia. Total parathyroidectomy causes hypoparathyroidism and durable remission of hypercalcemia. Some other features are not shared with PHPT and could support FHH as a distinct entity. These include onset of hypercalcemia in the first week of life, frequent persistence of hypercalcemia after subtotal parathyroidectomy, and hypocalciuria. The features supporting FHH as a form of PHPT are stronger than those favoring FHH as a distinct entity. Classifying FHH as an atypical form of PHPT represents compact nomenclature and supports current concepts of pathophysiology of FHH and PHPT. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.     

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium‐Sensing Receptor‐Mediated Signaling

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β‐catenin), the molecular basis of abnormal calcium sensing by calcium‐sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein‐coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3′,5′‐cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N‐terminally truncated human GPR64 is constitutively active and a 15–amino acid–long peptide C‐terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full‐length GPR64 physically interacts with CaSR and attenuates the CaSR‐mediated intracellular Ca²⁺ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

Development and Validation of a Novel Laboratory‐Specific Correction Equation for Total Serum Calcium and Its Association With Mortality Among Hemodialysis Patients

Conventional albumin‐corrected calcium is inaccurate in predicting ionized calcium, and hidden hypercalcemia, characterized as high ionized calcium with normal total calcium, is associated with higher mortality in hemodialysis patients. By using a national cohort of hemodialysis patients in the Unites States, a novel laboratory‐specific prediction equation composed of total calcium, albumin, and phosphorus was derived from 242 patients in the South Atlantic division (adjusted R² = 0.80 versus 0.71 for the conventional equation) and then validated among 566 patients in the other divisions (adjusted R² = 0.79 versus 0.68 for the conventional equation). Compared with the conventional equation, the novel equation showed a greater correlation with intact parathyroid hormone. Its relative performance against the conventional equation was consistent across subgroups based on medications related to calcium metabolism. The novel equation also had a higher sensitivity (57% versus 34%) and an equivalent specificity (99% versus 100%) against ionized hypercalcemia at a cut‐off value of 10.2 mg/dL. Sensitivity and specificity at 9.4 mg/dL was 94% and 76% (versus 87% and 82% for the conventional equation), respectively. A survival analysis in 87,779 incident hemodialysis patients showed that among patients who were categorized as having a high‐normal calcium status (ie, >9.4 to 10.2 mg/dL) by the conventional equation, there appeared a trend toward higher adjusted mortality risk across higher calcium status defined according to the novel equation. Meanwhile, the mortality risk was consistent across calcium strata defined according to the conventional equation within the categories defined by the novel equation. In conclusion, in comparison to the conventional equation, a novel laboratory‐specific correction equation derived for correction of total calcium performs significantly better in ascertaining hidden hypercalcemia in hemodialysis patients, and aids in identifying patients at higher risk for mortality. © 2016 American Society for Bone and Mineral Research.     

Forearm Bone Mineral Density in Familial Hypocalciuric Hypercalcemia and Primary Hyperparathyroidism: A Comparative Study

Studies have shown that cancellous bone is relatively preserved in primary hyperparathyroidism (PHPT), whereas bone loss is seen in cortical bone. Familial hypocalciuric hypercalcemia (FHH) patients seem to preserve bone mineral in spite of hypercalcemia and often elevated plasma parathyroid hormone (PTH). The objective of this study was to compare total and regional forearm bone mineral density (BMD) in patients with PHPT and FHH and to examine if differences can be used to separate the two disorders. We included 63 FHH, and 121 PHPT patients in a cross-sectional study. We performed dual-energy X-ray absorptiometry scans of the forearm, hip and lumbar spine and measured a number of biochemical variables. PTH patients had significantly lower Z-scores in all parts of the forearm compared to FHH. This was also the case after adjustment for body mass index. When stratifying for age, gender and PTH, T-scores were still significantly lower in PHPT patients than in FHH patients at the total, the mid and the ultradistal forearm, but not at the proximal 1/3 forearm. In a multiple regression analysis BMD Z-score was lower in PHPT compared to FHH at the total forearm, the mid forearm and the ultradistal forearm but not the proximal forearm when adjusting for biochemical variables including PTH, 1,25(OH)₂D and Ca²⁺. These observations support that inactivating mutations in the CASR gene in bone cells in FHH may protect against forearm bone loss. Differences between the two groups in total or regional forearm BMD were inferior to the calcium/creatinine clearance ratio as a diagnostic tool to separate FHH from PHPT.     

Beneficial effect of cinacalcet in a child with familial hypocalciuric hypercalcemia

We describe a child with familial hypocalciuric hypercalcemia (FHH) in whom the hypercalcemia seemed to interfere with tissue healing after tympanoplasty. Consequently, he was placed on cinacalcet (30 mg/day), changed after 2 weeks to 60 mg/day. The treatment resulted in a decrease in serum parathyroid hormone (PTH) from 148 to 32 pg/mL (normal 7–75) and ionized calcium from 1.48 to 1.23 mmol/L (1.13–1.34), as well as successful healing of the revised surgical scar. Over the 12-month treatment period no complications were noted. We conclude that cinacalcet may be considered a new, and currently the only, tool in treating children with symptomatic FHH.     

A Young Woman With Recurrent Gestational Hypercalcemia and Acute Pancreatitis Caused by CYP24A1 Deficiency

The CYP24A1 gene encodes a mitochondrial 24‐hydroxylase that inactivates 1,25(OH)₂D. Loss‐of‐function mutations in CYP24A1 cause hypercalcemia, nephrolithiasis and nephrocalcinosis. We describe a woman with CYP24A1 deficiency and recurrent gestational hypercalcemia. Her first pregnancy, at age 20, resulted with the intrauterine demise of twin fetuses. Postpartum, she developed severe hypercalcemia (14 mg/dL), altered mental status, and acute pancreatitis. Her PTH was suppressed (6 pg/mL) and her 1,25(OH)₂D was elevated (165 and 195 pg/mL on postpartum day 1 and 5, respectively). Between one and three months postpartum, her serum calcium decreased from 11.4 to 10.2 mg/dL while her 1,25(OH)₂D level decreased from 83 to 24 pg/mL. Her 24‐hour urine calcium was 277 mg. Six months postpartum, she became pregnant again. At 14 weeks, her albumin‐corrected calcium level was 10.4 mg/dL and her 1,25(OH)₂D level exceeded 200 pg/mL. To establish the diagnosis of CYP24A1 deficiency, we showed her 24,25(OH)₂D level to be undetectable (<2 ng/mL). Exon sequencing of the CYP24A1 gene revealed a homozygous, 8‐nucleotide deletion in exon 8, causing an S334V substitution and premature termination due to a frame shift (c.999_1006del, p.Ser334Valfs*9). To prevent hypercalcemia, she was advised to discontinue prenatal vitamins, avoid sun exposure and calcium‐rich foods, and start omeprazole and a calcium binder (250 mg K‐Phos‐neutral with meals). Despite these measures, both hypercalcemia (11.5 mg/dL) and acute pancreatitis recurred. Labor was induced and a healthy, normocalcemic boy was delivered. In the absence of lactation, maternal hypercalcemia resolved within 2 months. This report shows that CYP24A1‐deficient subjects may be normocalcemic at baseline. Hypercalcemia may be unmasked by pregnancy through the routine use of calciferol‐containing prenatal vitamins, increased 1‐alpha hydroxylation of VitD by the placenta and maternal kidney, and production of PTHrP by the uteroplacental unit. CYP24A1 deficiency should be considered in patients with unexplained vitamin D‐mediated hypercalcemia. © 2016 American Society for Bone and Mineral Research.     

The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

Increased Trabecular Volumetric Bone Mass Density in Familial Hypocalciuric Hypercalcemia (FHH) Type 1: A Cross-Sectional Study

Familial Hypocalciuric Hypercalcaemia (FHH) Type 1 is caused by an inactivating mutation in the calcium-sensing receptor (CASR) gene resulting in elevated plasma calcium levels. We investigated whether FHH is associated with change in bone density and structure. We compared 50 FHH patients with age- and gender-matched population-based controls (mean age 56 years, 69 % females). We assessed areal BMD (aBMD) by DXA-scans and total, cortical, and trabecular volumetric BMD (vBMD) as well as bone geometry by quantitative computed tomography (QCT) and High-Resolution peripheral-QCT (HR-pQCT). Compared with controls, FHH females had a higher total and trabecular hip vBMD and a lower cortical vBMD and hip bone volume. Areal BMD and HRpQCT indices did not differ except an increased trabecular thickness and an increased vBMD at the transition zone between cancellous and cortical bone in of the tibia in FHH. Finite element analyses showed no differences in bone strength. Multiple regression analyses revealed correlations between vBMD and P-Ca²⁺ levels but not with P-PTH. Overall, bone health does not seem to be impaired in patients with FHH. In FHH females, bone volume is decreased, with a lower trabecular volume but a higher vBMD, whereas cortical vBMD is decreased in the hip. This may be due to either an impaired endosteal resorption or corticalization of trabecular bone. The smaller total bone volume suggests an impaired periosteal accrual, but bone strength is not impaired. The findings of more pronounced changes in females may suggest an interaction between sex hormones and the activity of the CaSR on bone.     

PTH(1‐84) replacement therapy in hypoparathyroidism: A randomized controlled trial on pharmacokinetic and dynamic effects after 6 months of treatment

Untreated, hypoparathyroidism (hypoPT) is a state of hypocalcemia with inappropriately low plasma parathyroid hormone (PTH) levels and hyperphosphatemia. PTH administration normalizes plasma calcium and phosphate levels and reduces the doses of calcium and active vitamin D analogues needed. To develop an evidence‐based clinical algorithm to monitor hypoPT patients treated with recombinant human PTH (rhPTH[1‐84]) injected subcutaneously in the thigh, we performed a 24‐hour monitoring study of pharmacokinetic and pharmacodynamic effects in a group of 38 patients who had completed a 6‐month randomized study on effects of treatment with a fixed rhPTH(1‐84) dose of 100 µg/d or similar placebo as an add‐on to conventional treatment. PTH levels rose immediately, reaching a median peak level of 26.5 (interquartile range [IQR], 20.1–42.5) pmol/L 15 minutes following injection. Thereafter, levels gradually decreased until reaching predosing levels after 16 hours, with a plasma half‐life of 2.2 (1.7–2.5) hours. rhPTH(1‐84) changed the diurnal rhythms of ionized calcium levels and 1,25‐dihydroxyvitamin D (1,25[OH]₂D) levels, with rising levels following injection. Ionized calcium peaked after 7.0 (5.0–10.0) hours. Asymptomatic hypercalcemia was present in 71% of the rhPTH(1‐84)‐treated patients. Compared with placebo, 24‐hour urinary calcium, phosphate, and magnesium did not change, although the diurnal variation in renal excretion rates changed significantly in response to treatment. In conclusion, as a safety precaution, we recommend occasionally measuring calcium levels at approximately 7 hours after administration in order to reveal episodes of hypercalcemia. A 100‐µg daily dose of rhPTH(1‐84) appears to be too high in some patients, suggesting a need for a device allowing for individual dose adjustments. © 2013 American Society for Bone and Mineral Research.     

Hypercalcemia and Elevated Serum 1.25‐Dihydroxyvitamin D in an End‐stage Renal Disease Patient with Pulmonary Cryptococcosis

Hypercalcemia occurs relatively often in dialysis patients. The most common cause of hypercalcemia in dialysis patients is the conventional therapy with calcium and calcitriol. Besides, secondary hyperparathyroidism, low turnover bone diseases, and immobilization are also common causes of hypercalcemia in dialysis patients. Fungal infection associated with hypercalcemia has been infrequently reported. We describe a 71‐year‐old female woman with end‐stage renal disease and diabetes mellitus, who developed severe hypercalcemia. Pulmonary cryptococcosis, with increased concentration of serum 1,25‐dihydroxyvitamin D (1,25(OH)₂D), was diagnosed. Her serum concentration of calcium and 1,25(OH)₂D returned to normal after antifungal treatment. Thus, hypercalcemia was mediated by extrarenal overproduction of 1,25(OH)₂D in this patient.     

Parathyroid hormone signaling via Gαs is selectively inhibited by an NH2‐terminally truncated Gαs: Implications for pseudohypoparathyroidism

Pseudohypoparathyroid patients have resistance predominantly to parathyroid hormone (PTH), and here we have examined the ability of an alternative Gαs‐related protein to inhibit Gαs activity in a hormone‐selective manner. We tested whether the GNAS exon A/B‐derived NH₂‐terminally truncated (Tr) αs protein alters stimulation of adenylate cyclase by the PTH receptor (PTHR1), the thyroid‐stimulating hormone (TSH) receptor (TSHR), the β₂‐adrenergic receptor (β₂AR), or the AVP receptor (V2R). HEK293 cells cotransfected with receptor and full‐length (FL) Gαs ± Tr αs protein expression vectors were stimulated with agonists (PTH [10^?7 to 10^?9 M], TSH [1 to 100 mU], isoproterenol [10^?6 to 10^?8 M], or AVP [10^?6 to 10^?8 M]). Following PTH stimulation, HEK293 cells cotransfected with PTHR1 + FL Gαs + Tr αs had a significantly lower cAMP response than those transfected with only PTHR1 + FL Gαs. Tr αs also exerted an inhibitory effect on the cAMP levels stimulated by TSH via the TSHR but had little or no effect on isoproterenol or AVP acting via β₂AR or V2R, respectively. These differences mimic the spectrum of hormone resistance in pseudohypoparathyroidism type 1a (PHP‐1a) and type 1b (PHP‐1b) patients. In opossum kidney (OK) cells, endogenously expressing the PTHR1 and β₂AR, the exogenous expression of Tr αs at a level similar to endogenous FL Gαs resulted in blunting of the cAMP response to PTH, whereas that to isoproterenol was unaltered. A pseudopseudohypoparathyroid patient with Albright hereditary osteodystrophy harbored a de novo paternally inherited M1I Gαs mutation. Similar maternally inherited mutations at the initiation codon have been identified previously in PHP‐1a patients. The M1I αs mutant (lacking the first 59 amino acids of Gαs) blunted the increase in cAMP levels stimulated via the PTHR1 in both HEK293 and OK cells similar to the Tr αs protein. Thus NH₂‐terminally truncated forms of Gαs may contribute to the pathogenesis of pseudohypoparathyroidism by inhibiting the activity of Gαs itself in a GPCR selective manner. © 2011 American Society for Bone and Mineral Research     

PHEX 3′‐UTR c.*231A>G Near The Polyadenylation Signal Is a Relatively Common,Mild, American Mutation That Masquerades as Sporadic or X‐Linked Recessive Hypophosphatemic Rickets

Heritable forms of hypophosphatemic rickets (HR) include X‐linked dominant (XLH), autosomal recessive, and autosomal dominant HR (from deactivating mutations in PHEX, DMP1 or ENPP1, and activating mutations in FGF23, respectively). Over 30 years, we have cared for 284 children with HR. For those 72 deemed sporadic XLH, we preliminarily reported mutation analysis for 30 subjects. Eleven had PHEX mutations. However, the remaining 19 lacked readily identifiable defects in PHEX, DMP1, or FGF23. In 2008, a novel single‐base change near the polyadenylation (pA) signal in the 3′‐UTR of PHEX was identified in XLH by other investigators. This c.*231A > G mutation is 3‐bp upstream of the putative pA signal (AATAAA) in PHEX. Accordingly, we investigated whether this 3′‐UTR defect accounted for HR in any of these 19 sporadic XLH patients. PCR amplification and sequencing of their 3′‐UTR region showed the c.*231A > G mutation in four unrelated boys. Then, among an additional 22 of our 72 “sporadic” XLH patients, one boy and one girl were found to have the 3′‐UTR defect, totaling six patients. Among these 52 sporadic XLH patients with PHEX analysis, 36 were girls and 16 were boys; ie, a ～2:1 gender ratio consistent with XLH. However, finding five boys and only one girl with this 3′‐UTR mutation presented an unexplained gender bias (p = 0.02). Haplotyping for the five boys, all reportedly unrelated, showed a common core haplotype suggesting a founder. Five of their six mothers had been studied clinically and biochemically (three radiologically). Remarkably, the seemingly unaffected mothers of four of these boys carried the 3′‐UTR mutation. These healthy women had normal height, straight limbs, lacked the radiographic presentation of XLH, and showed normal or slight decreases in fasting serum Pi levels and/or TmP/GFR. Hence, PHEX c.*231A > G can masquerade as sporadic or X‐linked recessive HR. © 2014 American Society for Bone and Mineral Research.     

Calcitonin Receptor Plays a Physiological Role to Protect Against Hypercalcemia in Mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)₂D₃]‐induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.     

Cyclooxygenase‐2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line

Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase‐2 (COX‐2) induction and prostaglandin E₂ (PGE₂) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX‐2 induction and PGE₂ production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca²⁺ concentration ([Ca²⁺]_i), PGE₂ production, and COX‐2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification‐induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca²⁺]_i and inositol phosphate production in the cells. Acidification also induced COX‐2 induction, resulting in PGE₂ production. These proton‐induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for G_q/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/G_q/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX‐2 induction and subsequent PGE₂ production in response to acidic circumstances.     

Familial hypocalciuric hypercalcemia revealed by chondrocalcinosis

BackgroundCalcium pyrophosphate dihydrate crystal deposition disease (CPPD-CDD) has been associated to hypercalcemia. Familial hypocalciuric hypercalcemia (FHH) is a rare but important consideration in the differential diagnosis of hypercalcemia. This autosomal dominantly inherited condition is characterized by elevated plasma calcium levels, relative or absolute hypocalciuria, and normal to moderately elevated plasma PTH level. The disease is caused by inactivating mutations in the calcium-sensing receptor gene.Case reportWe describe a 77-year-old Italian man with arthritis secondary to CPPD-CDD and hypercalcemia. Clinical and biochemical data (s-Ca: 2.94 mmol/L; PTH: 5.9 pmol/L; 24 h urinary calcium: 69.6 mg; calcium/creatinine clearance: 0.004) suggested the diagnosis of FHH. Mild hypocalciuric hypercalcemia was also found in five of seven relatives confirming the diagnosis, of these one showed chondrocalcinosis.ConclusionsIt is important to screen for FHH using fractional urinary excretion of calcium in subjects with CPPD-CDD associated to hypercalcemia, this approach may prevent unnecessary parathyroidectomy.     

A semimechanistic model of the time‐course of release of PTH into plasma following administration of the calcilytic JTT‐305/MK‐5442 in humans

JTT‐305/MK‐5442 is a calcium‐sensing receptor (CaSR) allosteric antagonist being investigated for the treatment of osteoporosis. JTT‐305/MK‐5442 binds to CaSRs, thus preventing receptor activation by Ca²⁺. In the parathyroid gland, this results in the release of parathyroid hormone (PTH). Sharp spikes in PTH secretion followed by rapid returns to baseline are associated with bone formation, whereas sustained elevation in PTH is associated with bone resorption. We have developed a semimechanistic, nonpopulation model of the time‐course relationship between JTT‐305/MK‐5442 and whole plasma PTH concentrations to describe both the secretion of PTH and the kinetics of its return to baseline levels. We obtained mean concentration data for JTT‐305/MK‐5442 and whole PTH from a multiple dose study in U.S. postmenopausal women at doses of 5, 10, 15, and 20 mg. We hypothesized that PTH is released from two separate sources: a reservoir that is released rapidly (within minutes) in response to reduction in Ca²⁺ binding, and a second source released more slowly following hours of reduced Ca²⁺ binding. We modeled the release rates of these reservoirs as maximum pharmacologic effect (E_max) functions of JTT‐305/MK‐5442 concentration. Our model describes both the dose‐dependence of PTH time of occurrence for maximum drug concentration (T_max) and maximum concentration of drug (C_max), and the extent and duration of the observed nonmonotonic return of PTH to baseline levels following JTT‐305/MK‐5442 administration.