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Familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2) are due to loss- and gain-of-function mutations, respectively, of the GNA11 gene that encodes the G protein subunit Gα11, a signaling partner of the calcium-sensing receptor (CaSR). To date, four probands with FHH2-associated Gα11 mutations and eight probands with ADH2-associated Gα11 mutations have been reported. In a 10-year period, we identified 37 different germline GNA11 variants in >1200 probands referred for investigation of genetic causes for hypercalcemia or hypocalcemia, comprising 14 synonymous, 12 noncoding, and 11 nonsynonymous variants. The synonymous and noncoding variants were predicted to be benign or likely benign by in silico analysis, with 5 and 3, respectively, occurring in both hypercalcemic and hypocalcemic individuals. Nine of the nonsynonymous variants (Thr54Met, Arg60His, Arg60Leu, Gly66Ser, Arg149His, Arg181Gln, Phe220Ser, Val340Met, Phe341Leu) identified in 13 probands have been reported to be FHH2- or ADH2-causing. Of the remaining nonsynonymous variants, Ala65Thr was predicted to be benign, and Met87Val, identified in a hypercalcemic individual, was predicted to be of uncertain significance. Three-dimensional homology modeling of the Val87 variant suggested it was likely benign, and expression of Val87 variant and wild-type Met87 Gα11 in CaSR-expressing HEK293 cells revealed no differences in intracellular calcium responses to alterations in extracellular calcium concentrations, consistent with Val87 being a benign polymorphism. Two noncoding region variants, a 40bp-5'UTR deletion and a 15bp-intronic deletion, identified only in hypercalcemic individuals, were associated with decreased luciferase expression in vitro but no alterations in GNA11 mRNA or Gα11 protein levels in cells from the patient and no abnormality in splicing of the GNA11 mRNA, respectively, confirming them to be benign polymorphisms. Thus, this study identified likely disease-causing GNA11 variants in <1% of probands with hypercalcemia or hypocalcemia and highlights the occurrence of GNA11 rare variants that are benign polymorphisms. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism due to activating variants of the calcium-sensing receptor gene (CASR). Inherited or de novo activating variants of the CASR alter the set point for extracellular calcium, resulting in inadequate parathyroid hormone (PTH) secretion and inappropriate renal calcium excretion leading to hypocalcemia and hypercalciuria. Conventional therapy includes calcium and activated vitamin D, which can worsen hypercalciuria, resulting in renal complications. A systematic literature review, using published reports from 1994 to 2021, was conducted to catalog CASR variants, to define the ADH1 clinical spectrum, and to determine the effect of treatment on patients with ADH1. There were 113 unique CASR variants reported, with a general lack of genotype/phenotype correlation. Clinical data were available in 191 patients; 27% lacked symptoms, 32% had mild/moderate symptoms, and 41% had severe symptoms. Seizures, the most frequent clinical presentation, occurred in 39% of patients. In patients with blood and urine chemistries available at the time of diagnosis (n = 91), hypocalcemia (99%), hyperphosphatemia (59%), low PTH levels (57%), and hypercalciuria (34%) were observed. Blood calcium levels were significantly lower in patients with severe symptoms compared with asymptomatic patients (6.8 ± 0.7 versus 7.6 ± 0.7 mg/dL [mean ± SD]; p < 0.0001), and the age of presentation was significantly lower in severely symptomatic patients (9.1 ± 15.0 versus 19.3 ± 19.4 years; p < 0.01). Assessments for complications including nephrocalcinosis, nephrolithiasis, renal impairment, and brain calcifications in 57 patients on conventional therapy showed that 75% had at least one complication. Hypercalciuria was associated with nephrocalcinosis, nephrolithiasis, renal impairment, or brain calcifications (odds ratio [OR] = 9.3; 95% confidence interval [CI] 2.4-37.2; p < 0.01). In 27 patients with urine calcium measures before and after starting conventional therapy, the incidence of hypercalciuria increased by 91% (p < 0.05) after therapy initiation. ADH1 is a condition often associated with severe symptomatology at presentation with an increase in the risk of renal complications after initiation of conventional therapy. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain-of-function mutations of the calcium-sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca 2+ i ), and ERK and p38 MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration-dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well-tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half-maximally activated (EC 50 ) at lower concentrations of extracellular calcium (Ca 2+ for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose-dependent manner, and thus, serves as proof-of-concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.o ) compared to wild-type (WT) CaR; NPSP795 exposure increased the EC 50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose-dependent manner, and thus, serves as proof-of-concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)

The role of the calcium-sensing receptor (CaSR) as a regulator of parathyroid hormone secretion is well established, but its function in bone is less well defined. In an effort to elucidate the CaSR's skeletal role, bone tissue and material characteristics from patients with autosomal dominant hypocalcemia (ADH), a genetic form of primary hypoparathyroidism caused by CASR gain-of-function mutations, were compared to patients with postsurgical hypoparathyroidism (PSH). Bone structure and formation/resorption indices and mineralization density distribution (BMDD), were examined in transiliac biopsy samples from PSH (n = 13) and ADH (n = 6) patients by histomorphometry and quantitative backscatter electron imaging, respectively. Bone mineral density (BMD by DXA) and biochemical characteristics were measured at the time of the biopsy. Because both study groups comprised children and adults, all measured biopsy parameters and BMD outcomes were converted to Z-scores for comparison. Histomorphometric indices were normal and not different between ADH and PSH, with the exception of mineral apposition rate Z-score, which was higher in the ADH group. Similarly, average BMD Z-scores were normal and not different between ADH and PSH. Significant differences were observed for the BMDD: average Z-scores of mean and typical degree of mineralization (CaMean, CaPeak, respectively) were lower (p = 0.02 and p = 0.03, respectively), whereas the heterogeneity of mineralization (CaWidth) and percentage of lower mineralized areas (CaLow) were increased in ADH versus PSH (p = 0.01 and p = 0.002, respectively). The BMDD outcomes point toward a direct, PTH-independent role of the CaSR in the regulation of bone mineralization. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

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 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Metabolic and bone effects were investigated in growing (G, n = 45) and mature (M, n = 45) rats fed a high-fat/high-sucrose diet (HFS) isocaloric to the chow diet of controls (C, n = 30 per group). At week 19, a subset of 15 rats in each group (HFS or C, at both ages) was analyzed. Then one-half of the remaining 30 HFS rats in each groups continued HFS and one-half were shifted to C until week 27. Although no serum or bone marrow inflammation was seen, HFS increased visceral fat, serum leptin and insulin at week 19 and induced further alterations in lipid profile, serum adiponectin, and TGFβ1, TIMP1, MMP2, and MMP9, suggesting a prediabetic phenotype and cardiovascular dysfunction at week 27 more pronounced in M than G. These events were associated with dramatic reduction of osteoclastic and osteoid surfaces with accelerated mineralizing surfaces in both HFS age groups. Mineral metabolism and its major regulators were disturbed, leading to hyperphosphatemia and hypocalcemia. These changes were associated with bone alterations in the weight-bearing tibia, not in the non-weight-bearing vertebra. Indeed in fat rats, tibia trabecular bone accrual increased in G whereas loss of trabecular bone in M was alleviated. At diaphysis cortical porosity increased in G and even more in M at week 27. After the diet switch, metabolic and bone cellular disturbances fully reversed in G, but not in M. Trabecular benefit of the obese was preserved in both age groups and in M the age-related bone loss was even lighter after the diet switch than in prolonged HFS. At the diaphysis, cortical porosity normalized in G but not in M. Hypocalcemia in G and M was irreversible. Thus, the mild metabolic syndrome induced by isocaloric HFS is able to alter bone cellular activities and mineral metabolism, reinforce trabecular bone, and affect cortical bone porosity in an irreversible manner in older rats., (© 2015 American Society for Bone and Mineral Research.)

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 Ca(2+) 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.)

Kenny-Caffey syndrome (KCS) is a rare dysmorphologic syndrome characterized by proportionate short stature, cortical thickening and medullary stenosis of tubular bones, delayed closure of anterior fontanelle, eye abnormalities, and hypoparathyroidism. The autosomal dominant form of KCS (KCS type 2 [KCS2]) is distinguished from the autosomal recessive form of KCS (KCS type 1 [KCS1]), which is caused by mutations of the tubulin-folding cofactor E (TBCE) gene, by the absence of mental retardation. In this study, we recruited four unrelated Japanese patients with typical sporadic KCS2, and performed exome sequencing in three patients and their parents to elucidate the molecular basis of KCS2. The possible candidate genes were explored by a de novo mutation detection method. A single gene, FAM111A (NM&#95;001142519.1), was shared among three families. An identical missense mutation, R569H, was heterozygously detected in all three patients but not in the unaffected family members. This mutation was also found in an additional unrelated patient. These findings are in accordance with those of a recent independent report by a Swiss group that KCS2 is caused by a de novo mutation of FAM111A, and R569H is a hot spot mutation for KCS2. Although the function of FAM111A is not known, this study would provide evidence that FAM111A is a key molecule for normal bone development, height gain, and parathyroid hormone development and/or regulation., (© 2014 American Society for Bone and Mineral Research.)

Unlabelled: To define the molecular pathogenesis of severe postnatal hypocalcemia in monozygotic twin sisters, we sequenced their CaR gene and identified a missense mutation, K29E. Expression of the mutant receptor in vitro showed a marked increase in Ca2+ sensitivity explaining the observed phenotype. Additional mutagenesis studies lead us to speculate concerning a novel mechanism whereby the K29E mutation may lead to receptor activation., Introduction: Activating mutations of the Ca(2+)-sensing receptor (CaR) gene have been identified in subjects with autosomal dominant hypocalcemia. Study of such mutations has provided insight into the mechanism of activation of the CaR., Materials and Methods: We performed biochemical and molecular genetic studies on monozygotic twin sisters who presented with early postnatal hypocalcemia and on their unaffected sister and parents. Functional characterization of mutant CaRs transfected in HEK-293 cells included immunoblots to monitor protein expression and Ca2+ stimulation of phosphoinositide hydrolysis to measure Ca2+ sensitivity., Results: We identified a K29E missense mutation in the twin sisters but not in their parents or unaffected sister. The K29E mutant CaR showed a marked increase in Ca2+ sensitivity, including when it was co-transfected with wildtype CaR cDNA, consistent with a dominant effect. Substitution of K29 by aspartate equivalently increased CaR sensitivity, whereas conservative substitution by arginine did not., Conclusions: Severe postnatal hypocalcemia in the twin sisters was caused by a de novo germline activating mutation. In a model of the Venus flytrap-like domain of the extracellular amino-terminus of the CaR, K29 is located close to a peptide loop, "loop 2," that forms part of the dimer interface and is the site of 10 of the previously reported naturally occurring activating CaR mutations. We speculate that K29E increases Ca2+ sensitivity of the CaR by disrupting a salt bridge between K29 and an acidic residue in loop 2 and thereby changes the normal structure of loop 2 that maintains the CaR in its inactive conformation.

We report a novel missense mutation N124K in the extracellular calcium receptor (CaR) identified in two related subjects with the phenotypic features of autosomal dominant hypocalcemia (ADH). Expression of the N124K mutant receptor created by site-directed mutagenesis and transfected into HEK-293 cells was comparable with that of the wild-type (WT) receptor and two other mutant receptors N118K and L125P identified in subjects with ADH. Functional characterization by the extracellular Ca2+ ion ([Ca2+]0)-stimulated phosphoinositide (PI) hydrolysis in transfected HEK-293 cells showed that the N124K mutant receptor was left-shifted in Ca2+ sensitivity. This biochemical gain-of-function is comparable with that seen in other missense mutations of the CaR identified in subjects with ADH. We tested a series of missense substitutions (R, Q, E, and G) in addition to K for N124 and found that only the N124K mutation and to a much lesser extent N124R caused a left shift in Ca2+ sensitivity. Thus, a specific substitution, not merely a mutation of the N124 residue, is required for receptor activation. The N124K mutation is one of eight naturally occurring mutations in subjects with ADH identified in a short segment A116-C129 of the CaR extracellular domain (ECD). We present a hypothesis to explain receptor activation by mutations in this region based on the recently described three-dimensional structure of the related metabotropic glutamate type 1 receptor (mGluR1).

Mutations in the vitamin D receptor (VDR) gene have been shown to cause hereditary vitamin D-resistant rickets (HVDRR). The patient in this study is a young French-Canadian boy with no known consanguinity in his family. The child exhibited the clinical characteristics of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-dihydroxyvitamin D (1,25(OH)2D) levels as well as total alopecia. Fibroblasts were cultured from a skin biopsy of the patient and used to assess the VDR. Northern blot analysis showed that a normal size VDR transcript was expressed; however, [3H]1,25(OH)2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Total resistance to 1,25(OH)2D3 was demonstrated by the failure of the cultured fibroblasts to induce the transcription of the 25-hydroxyvitamin D-24-hydroxylase gene when treated with high concentrations of 1,25(OH)2D3. Analysis of the DNA sequence revealed a unique C to T base change corresponding to nucleotide 218 of the VDR cDNA. This single base substitution changes the codon for arginine (CGA) to an opal stop codon (TGA), resulting in the truncation of the VDR at amino acid 30. The Arg30stop mutation prematurely terminates translation and deletes 398 amino acids including most of the zinc fingers as well as the entire ligand-binding domain. Restriction fragment length polymorphism analysis of a DdeI restriction site created by the mutation showed that the parents were heterozygous for the mutant allele. In conclusion, the Arg30stop mutation truncates the VDR and leads to a hormone-resistant condition which is the molecular basis of HVDRR in this patient.

High calcium leads to the secretion of calcitonin, and the administration of 1,25-dihydroxyvitamin D3 leads to a decreased transcription of the calcitonin gene. We now report the effect of chronic hypercalcemia, hypocalcemia, and vitamin D deficiency on calcitonin gene expression in vivo in the rat. Hypercalcemia was created by calcium infusions for 6 h, a high-calcium diet given to weanling rats for 3 weeks, and the transplantation of the Walker carcinosarcoma 256 cell line. Despite serum calcium as high as 22 mg/dl, there was no difference in calcitonin mRNA levels among these rats. The control genes studied, actin and somatostatin, which is specific for C cells in the thyroparathyroid tissue, also did not differ among the different groups of rats. Injected 1,25-(OH)2D3 decreased calcitonin mRNA levels at 6 h, as previously reported. Hypocalcemia, created by feeding diets deficient in calcium and vitamin D to weanling rats for 3 weeks, had no effect on calcitonin mRNA levels, in contrast to the large increases in PTH mRNA levels. These results demonstrate that calcitonin gene expression in vivo in the rat is regulated by administered 1,25-(OH)2D3 but not by changes in serum calcium.