Homozygous Calcium-Sensing Receptor Polymorphism R544Q Presents as Hypocalcemic Hypoparathyroidism

Homozygous Calcium-Sensing Receptor Polymorphism R544Q Presents as Hypocalcemic Hypoparathyroidism Abstract Context Autosomal dominant hypocalcemia type 1 (ADH1) is caused by heterozygous activating mutations in the calcium-sensing receptor gene (CASR). Whether polymorphisms that are benign in the heterozygous state pathologically alter receptor function in the homozygous state is unknown. Objective To identify the genetic defect in an adolescent female with a history of surgery for bilateral cataracts and seizures. The patient has hypocalcemia, hyperphosphatemia, and low serum PTH level. The parents of the proband are healthy. Methods Mutation testing of PTH, GNA11, GCM2, and CASR was done on leukocyte DNA of the proband. Functional analysis in transfected cells was conducted on the gene variant identified. Public single nucleotide polymorphism (SNP) databases were searched for the presence of the variant allele. Results No mutations were identified in PTH, GNA11, and GCM2 in the proband. However, a germline homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR was identified. Both parents are heterozygous for the variant. The variant allele frequency was near 0.1% in SNP databases. By in vitro functional analysis, the variant was significantly more potent in stimulating both the Ca2+i and MAPK signaling pathways than wild type when transfected alone (P < 0.05) but not when transfected together with wild type. The overactivity of the mutant CaSR is due to loss of a critical structural cation-π interaction. Conclusions The patient’s hypoparathyroidism is due to homozygosity of a variant in the CASR that normally has weak or no phenotypic expression in heterozygosity. Although rare, this has important implications for genetic counseling and clinical management. Congenital or familial isolated hypoparathyroidism may present in an autosomal dominant or recessive fashion and can be due to mutations in the calcium-sensing receptor gene (CASR), glial cells missing-2 gene (GCM2), G protein α11 gene (GNA11), or parathyroid hormone gene (PTH) (1). Gain-of-function mutations in the CASR (MIM#601199) have been identified in a number of families with autosomal dominant hypocalcemia type 1 (ADH1) (MIM#601198). In the parathyroid gland, the activated CaSR (the calciostat) suppresses PTH secretion, and in the kidney, in some cases it induces hypercalciuria that may contribute to the hypocalcemia (2, 3). The CaSR is a member of G protein‒coupled receptor family C. The mature human protein has an extracellular domain (ECD) of 593 amino acids, a 250‒amino acid transmembrane domain (TMD), and a 216‒amino acid intracellular tail (4, 5). The CaSR functions as a dimer. The ECD of each CaSR monomer has a bilobed Venus flytrap‒like (VFT) domain followed by a cysteine-rich domain and a linker region before the TMD (5, 6). Recently, the crystal structure of the ECD of the CaSR was elucidated to provide insights into binding to the VFT of Ca2+ and Mg2+ and amino acids and subsequent conformational changes leading to activation (7, 8). Of the ADH1 mutations identified to date, >65 are located in the ECD, and three-quarters of these are found at the dimer interface, with the remainder at the ligand-binding or other sites (3). In a study in which the structure of the entire ECD was determined, the importance of the cysteine-rich domain in contributing to the extended dimerization interface upon ligand binding was revealed (7). Several signaling pathways are activated by the CaSR (9). The stimulated CaSR couples to Gq/11, causing phospholipase C‒mediated production of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the latter of which causes intracellular Ca2+ mobilization from endoplasmic reticulum stores. It also activates MAPK family members (9). In some cases, in vitro functional testing of mutant CaSRs has been critical in correlating the clinical presentation with the relative overactivity of particular ADH1 mutations (10, 11). In the current study, we report the identification of a rare missense variant in the CaSR cysteine-rich domain that, while not clinically expressed in the heterozygous state, came to our attention in an individual who is homozygous for the variant. Our study also revealed a particular structural feature of the CaSR—a cation-π interaction—that when lost makes the receptor more responsive to extracellular calcium. Patient and Methods Case report The proband is a 20-year-old female patient with hypoparathyroidism (II-1; Fig. 1; Table 1). She was born a healthy baby at 40 weeks (weight, 3800 g; length, 52 cm), and her perinatal history was uneventful. Her psychomotor development was normal, with no evidence of tetany or seizures. She was breastfed until 2.5 months of age and was supplemented with 25-hydroxyvitamin D until the age of 1 year. At age 16 years, she underwent surgery for bilateral cataracts. She was evaluated at 18 years of age for an episode of seizures without any intercurrent illness. Laboratory analysis showed hypocalcemia, 4.4 mg/dL [normal range (NR) = 8.4 to 10.2 mg/dL], hyperphosphatemia, 8.7 mg/dL (NR = 2.3 to 4.7 mg/dL), undetectable serum PTH level, <2.5 pg/mL (NR = 12 to 65 pg/mL), and hypocalciuria, 0.02 g/L (NR = 0.05 to 1.15 g/L) (Table 1). At her first visit to our clinic at 20 years of age, she was under calcium (1200 mg/d) and calcitriol (0.5 µg/d) treatment. Her serum calcium level was 6.6 mg/dL, with hyperphosphatemia (8.2 mg/dL) and normal levels of 25-hydroxyvitamin D (32 ng/mL; NR = 9 to 45 ng/mL) (Table 1). She did not demonstrate paresthesia, cramps, or tetany, and Chvostek and Trousseau signs were negative. Electrocardiography revealed a prolonged QTc interval (451 ms). Brain CT showed basal ganglia calcification. Nephrocalcinosis was excluded by renal ultrasonography. Polyglandular autoimmune syndrome was excluded on laboratory and clinical grounds, and patient serum was negative for CaSR antibodies. Figure 1. View largeDownload slide Detection of a variant in the CASR gene. (A) Pedigree of family with ADH1 and CASR variant: open symbol, unaffected; filled symbol, affected. Proband is indicated by the arrow. CASR genotypes; +, wild-type allele; R554Q, variant allele. (B) Direct sequence analysis of CASR exon 6 genomic DNA amplicon revealed heterozygosity in parents I-1 and I-2 and homozygosity in proband II-1 for the c.1631G>A; p.R544Q variant. (C) The CaSR protein sequences from diverse species were aligned as described in Materials and Methods. The residue corresponding to the R544Q variant is boxed as are the conserved C542 and C546 residues in the CaSR cysteine-rich domain. (D) Western blot analysis of extracts of HEK293 cells transiently transfected with vector (pcDNA3.1), wild type (WT), or variant (R544Q) c-Myc‒tagged CaSR cDNAs. (Di) Recombinant proteins stained with c-Myc monoclonal antibody 9E10. (Dii) Endogenous proteins stained with a tubulin antibody. Bo, bovine; Ch, chicken; Hu, human; Mo, mouse; Ra, Rat; Ze; zebrafish. Figure 1. View largeDownload slide Detection of a variant in the CASR gene. (A) Pedigree of family with ADH1 and CASR variant: open symbol, unaffected; filled symbol, affected. Proband is indicated by the arrow. CASR genotypes; +, wild-type allele; R554Q, variant allele. (B) Direct sequence analysis of CASR exon 6 genomic DNA amplicon revealed heterozygosity in parents I-1 and I-2 and homozygosity in proband II-1 for the c.1631G>A; p.R544Q variant. (C) The CaSR protein sequences from diverse species were aligned as described in Materials and Methods. The residue corresponding to the R544Q variant is boxed as are the conserved C542 and C546 residues in the CaSR cysteine-rich domain. (D) Western blot analysis of extracts of HEK293 cells transiently transfected with vector (pcDNA3.1), wild type (WT), or variant (R544Q) c-Myc‒tagged CaSR cDNAs. (Di) Recombinant proteins stained with c-Myc monoclonal antibody 9E10. (Dii) Endogenous proteins stained with a tubulin antibody. Bo, bovine; Ch, chicken; Hu, human; Mo, mouse; Ra, Rat; Ze; zebrafish. Table 1. Biochemical Characteristics of Family Members Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Abbreviation: 25(OH)D, 25-hydroxyvitamin D. a Normal ranges in parentheses. b Before treatment with calcium and calcitriol. c On treatment with calcium and calcitriol. View Large Table 1. Biochemical Characteristics of Family Members Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Abbreviation: 25(OH)D, 25-hydroxyvitamin D. a Normal ranges in parentheses. b Before treatment with calcium and calcitriol. c On treatment with calcium and calcitriol. View Large Both parents (individuals I-1 and I-2; Fig. 1; Table 1) are clinically healthy with normal serum calcium and phosphate levels and normal fractional excretion of calcium. The parents are unaware of consanguinity, although they do share identical haplotypes at the CASR locus (Supplemental Table 1), which suggests they may have a common ancestor. DNA sequence analysis Written informed consent was obtained for blood collection and analyses of relevant genes in the proband and her parents. In brief, the analysis of the CASR, GNA11, PTH, and GCM2 genes involved genomic DNA extraction from peripheral blood leukocytes, PCR amplification of the gene exons including the adjoining splice junctions (primer sequences are available on request), and direct sequencing of the PCR products. Publicly accessible databases were examined for the presence of the identified sequence variant: the Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org/), representing exomes of 60,706 unrelated individuals (12); the Genome Aggregation Database (gnomad.broadinstitute.org/), representing 277,238 alleles (from ExAc and other sources); the ClinVar (www.ncbi.nlm.nih.gov/clinvar/), an archive of reports of the relationships among human variations and phenotypes (13); and the Human Gene Mutation Database (www.hgmd.cf.ac.uk/ac/index.php/), which collates known (published) gene lesions responsible for human inherited disease. Protein sequence alignment and three-dimensional modeling of CaSR structure Protein sequences of CaSR orthologues were aligned using ClustalOmega (www.ebi.ac.uk/Tools/msa/clustalo/) (14). The crystal structures of the human CaSR ECD in the inactive (5K5T) and active (5K5S) conformations (7) were obtained from the Protein Data Bank at www.rcsb.org (15) and analyzed using UCSF Chimera (16). The mutation tool that replaces one amino acid with another while selecting the rotamer with the highest χ2 statistic without directly affecting neighboring amino acids was used to replace R544 with Q544. Interactions between R544 and other amino acids within a 10Å radius were evaluated for inactive and active states of the receptor. Cation-π interactions and their energies were predicted with the CAPTURE program (17). The PyMOL Molecular Graphics System (PyMOL v1.8.6.2) (www.pymol.org/) was used to visualize protein structure. Site-directed mutagenesis and transfection Mutations were introduced into a c-Myc‒tagged human CaSR cDNA in pcDNA3.1 (18). The correctness of the constructs was confirmed by sequencing. Human embryonic kidney (HEK) 293 cells were transfected with c-Myc‒tagged human CaSR cDNAs, WT or mutant (19). Western blot analysis Proteins were isolated from HEK293 cells transiently transfected with WT or mutant c-Myc‒tagged CaSR cDNAs and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis through a 6% to 10% linear gradient gel, followed by Western blotting (19). Functional analysis of CaSRs Increased intracellular Ca2+ activates the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway. An NFAT promoter‒luciferase (Luc) assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca2+ (11). HEK293 cells were plated at 1 × 106 cells per well in six-well dishes in DMEM (#D9785; Sigma-Aldrich, Oakville, ON, Canada) plus 10% fetal bovine serum and cultured overnight to 70% confluency. Cells were then transiently cotransfected with 1.0 μg of WT or mutant CaSR constructs or empty vector or with 0.5 μg of WT and 0.5 μg of mutant CaSR, 0.5 μg of pGL3-NFAT-Luc reporter vector (plasmid 17870; Addgene, Cambridge, MA), and 0.5 μg of pRSV-Gal β-galactosidase vector using 20 μL per well PolyFect Transfection Reagent (Qiagen, Valencia, CA). After 16 hours, cells were serum starved in DMEM containing 0.5 mM of CaCl2, 0.5 mM of MgCl2 for 8 hours and treated with CaCl2 concentrations from 0.5 to 20 mM for 16 hours. Cells were then washed twice with PBS and lysed in 100 μL per well passive lysis buffer (Promega, Madison, WI). Luc activity was measured in a FLUOstar Optima Luminometer (BMG Labtech, Guelph, ON, Canada) and expressed relative to β-galactosidase activity. The activation of the ERK/MAPK pathway by the CaSR constructs in response to extracellular Ca2+ was made with pGL4.33 (Promega), a serum response element (SRE) promoter‒Luc reporter vector (10, 20). HEK293 cells were plated in six-well plates and grown to 70% confluency and transiently transfected using 20 μL per well PolyFect Transfection Reagent (Qiagen) with 1.0 μg of WT or mutant CaSR or empty vector or with 0.5 μg of WT and 0.5 μg of mutant CaSR, 0.5 μg of pGL4.33, and 0.5 μg of pRSV-Gal β-galactosidase vector. The following day, cells were serum-starved in DMEM containing 0.5 mM CaCl2, 0.5 mM MgCl2 for 8 hours and cultured in various concentrations of CaCl2 ranging from 0.5 to 20 mM for 16 hours. Cell extracts were prepared, and relative Luc activity was determined. A heterozygous CASR c.1810G>A; p.E604K mutation has been described in affected members of three independent families with ADH1 (21–23). In vitro functional studies have documented that CaSR E604K is properly expressed at the plasma membrane of transfected cells and is more active than WT CaSR in extracellular Ca2+ signaling pathway assays (21). We prepared a CaSR E604K expression construct and used it in functional assays as a reference for the relative activities obtained with the CaSR R544Q construct. Effect of negative allosteric modulator (NPS 2143) Functional assays were conducted as described previously, with 3.0 or 5.0 mM of Ca2+ as these concentrations bracket the EC50 value for the dose-response curve of the wild-type CaSR. At the time of changing the cell media to these Ca2+ concentrations, 50, 100, or 500 nM of NPS 2143 (synthesized in-house) in 0.1% dimethyl sulfoxide (or vehicle only) was added, and the incubation continued for 16 hours before harvest, cell lysis, and Luc assay. Statistical analysis Data are mean ± SEM. Results shown are representative of assays repeated three times. Nonlinear regression of concentration-response curves was performed with GraphPad Prism (La Jolla, CA) using the normalized response at each extracellular Ca2+ concentration [(Ca2+)o] for the determination of EC50 [i.e., (Ca2+)o required for 50% of the maximal response]. Statistical comparisons were made with the Mann-Whitney U test. A P value < 0.05 was considered significant. Supplemental methods CaSR intact cell surface ELISA, crude plasma membrane preparation and western blotting, and IP-One Assay (Cisbio, Burlington, ON, Canada) are described in online Supplemental Data. Results Identification of CASR variant No mutations were identified in the PTH, GNA11, and GCM2 genes in the germline DNA of the proband (II-1; Fig. 1A). However, a homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR gene was identified (Fig. 1B). Both parents (I-1 and I-2; Fig. 1A) were heterozygous for the variant (Fig. 1B). An arginine at position 544 is highly conserved among species (Fig. 1C). The R544Q variant does appear in single nucleotide polymorphism databases at a frequency of 0.08% (ExAC) and 0.09% (Genome Aggregation Database) (Table 2). The variant appears in the ClinVar database under eight entries, with descriptions covering both hypercalcemic (hyperparathyroid) and hypocalcemic (hypoparathyroid) conditions (Table 2). The clinical significance of these entries is given as likely benign or uncertain. A published report documents heterozygosity of the R544Q variant in an individual with familial hypocalciuric hypercalcemia (24). All entries in databases and reports are for R544Q as a heterozygote. Table 2. R544Q Variants in Public Databasesa Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Abbreviations: FHH1, familial hypocalciuric hypercalcemia type 1; gnomAD, Genome Aggregation Database. a All variants are heterozygotes. b Single entries of c.1631G>T; R544L and c.1630C>T; R544Ter occur in the databases. c In ExAc and gnomAD databases, R554Q is identified (rather than R544Q) as a different splice variant of the CaSR mRNA is used as the reference that encodes 10 extra amino acids (1088 rather than 1078), increasing the number by 10 after amino acid 536. Major form: NM_000388.3, NP_000379.2, 1078 amino acids; minor form: NM_001178065.1, NP_001171536.1, 1088 amino acids. View Large Table 2. R544Q Variants in Public Databasesa Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Abbreviations: FHH1, familial hypocalciuric hypercalcemia type 1; gnomAD, Genome Aggregation Database. a All variants are heterozygotes. b Single entries of c.1631G>T; R544L and c.1630C>T; R544Ter occur in the databases. c In ExAc and gnomAD databases, R554Q is identified (rather than R544Q) as a different splice variant of the CaSR mRNA is used as the reference that encodes 10 extra amino acids (1088 rather than 1078), increasing the number by 10 after amino acid 536. Major form: NM_000388.3, NP_000379.2, 1078 amino acids; minor form: NM_001178065.1, NP_001171536.1, 1088 amino acids. View Large Expression of wild-type and variant CaSRs in HEK293 cells The CaSR 544Q variant was expressed at equivalent levels and exhibited the same pattern of molecular species as CaSR WT: the core glycosylated (immature) 140-kDa species and the mature, fully glycosylated 160-kDa species (Fig. 1D). High molecular mass forms (∼300 kDa), likely to be dimers, were seen equally for the variant and WT receptors (Fig. 1D). The data show the variant achieved proper maturation and was appropriately trafficked to the plasma membrane under a variety of conditions (Supplemental Figs. 1 and 2). Responsiveness of the CaSR R544Q variant to extracellular calcium The ability of the variant receptor to respond to extracellular calcium relative to the WT receptor was assessed in three different assays. In the NFAT-Luc assay that monitors changes in intracellular Ca2+, the WT CaSR showed an EC50 of 4.3 ± 0.16 mM Ca2+ (Fig. 2A). The variant R544Q showed a significant (P < 0.05) leftward shift in its concentration-response curve (EC50, 3.6 ± 0.23 mM) relative to that of WT. When equal amounts of WT and variant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.95 ± 0.19 mM) to a position intermediate to, but not significantly different from, that of WT alone [not significant (NS)] and the variant alone (NS). Figure 2. View largeDownload slide (A) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either wild-type alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the NFAT-Luc promoter reporter construct. (B) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the SRE-Luc promoter reporter construct. (C) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), E604K and WT together (E604K/WT), or E604K alone (E604K) and the SRE-Luc promoter reporter construct. (D) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the NFAT-Luc promoter reporter construct. (E) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the SRE-Luc promoter reporter construct. (F) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or F539A and the NFAT-Luc promoter reporter construct. For each panel (A–F), values shown are the means of three replicates (SEM ≤45, not shown). Figure 2. View largeDownload slide (A) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either wild-type alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the NFAT-Luc promoter reporter construct. (B) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the SRE-Luc promoter reporter construct. (C) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), E604K and WT together (E604K/WT), or E604K alone (E604K) and the SRE-Luc promoter reporter construct. (D) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the NFAT-Luc promoter reporter construct. (E) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the SRE-Luc promoter reporter construct. (F) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or F539A and the NFAT-Luc promoter reporter construct. For each panel (A–F), values shown are the means of three replicates (SEM ≤45, not shown). In the SRE-Luc assay that monitors changes in ERK/MAPK activity, the WT CaSR cDNA showed an EC50 of 4.1 ± 0.16 mM Ca2+ (Fig. 2B). The variant R544Q showed a significant (P < 0.05) leftward shift in its concentration-response curve (EC50, 3.3 ± 0.18 mM) relative to that of WT. When equal amounts of WT and variant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.7 ± 0.19 mM) to a position intermediate to, but not significantly different from, that of WT alone (NS) and the variant alone (NS). Hence, in both types of promoter-Luc assay, the variant alone had a significantly greater potency than WT CaSR alone but not when cotransfected with WT CaSR. The ADH1 CaSR E604K mutant was used as a reference control in the SRE-Luc assay. The WT CaSR showed an EC50 of 4.1 ± 0.16 mM Ca2+ (Fig. 2C). The mutant E604K showed a significant (P < 0.01) leftward shift in its concentration-response curve (EC50, 3.1 ± 0.13 mM) relative to that of WT. When equal amounts of WT and mutant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.6 ± 0.14 mM) to a position intermediate to, but significantly different from, that of WT alone (P < 0.05) and the mutant alone (P < 0.05). Similar findings were obtained in the NFAT-Luc assay (Supplemental Fig. 3). Hence, the E604K mutant alone had a significantly greater potency than WT CaSR alone and also when cotransfected with WT CaSR in contrast to CaSR R544Q (Fig. 2A and 2B). In the IP-One assay that monitors changes in inositol phosphate, similar findings were obtained for the relative potencies of WT alone, R544Q/WT together, and R544Q alone (Supplemental Fig. 4A) and WT alone, E604K/WT together, and E604K alone (Supplemental Fig. 4), as in the NFAT-Luc and SRE-Luc assays. Further engineered mutants were evaluated as follows. An R544K mutant demonstrated potency identical to that of WT in both NFAT-Luc (Fig. 2D) and SRE-Luc (Fig. 2E) assays. An F539A mutant demonstrated enhanced potency similar to that of the R544Q variant in both NFAT-Luc (Fig. 2F) and SRE-Luc (Supplemental Fig. 5A) assays. The F539L variant demonstrated enhanced potency similar to that of the R544Q variant in both NFAT-Luc (Supplemental Fig. 5B) and SRE-Luc (Supplemental Fig. 5C) assays. A lower dose of NPS 2143 is required to restore the activity of R544Q to that of WT CaSR relative to an ADH1 CaSR mutant The calcilytic NPS 2143 has the ability to decrease the responsiveness of overactive mutant CaSRs to extracellular Ca2+ (10, 25). The relative amount of NPS 2143 required to restore the activity of a particular ADH1 CaSR mutant to that of WT CaSR indicates its relative potency as a gain-of-function mutant. Relative suppression of the signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143 was evaluated. A functional NFAT-Luc assay was conducted at [Ca2+]o of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143 (Fig. 3). Although 50 nM NPS 2143 was sufficient to restore the activity of CaSR R544Q to that of CaSR WT in the absence of the calcilytic at both [Ca2+]o of 3 and 5 mM, CaSR E604K remained highly significantly overactive at this particular NPS 2143 concentration (Fig. 3A). Increasing concentrations (100 and 500 nM) of NPS 2143 reduced the activities of both R544Q and E604K further while maintaining the relationship of much less activity for the R544Q variant relative to the E604K mutant at each particular NPS 2143 concentration tested (Fig. 3A). Similar findings were obtained in SRE-Luc assays (Fig. 3B). Figure 3. View largeDownload slide Relative suppression of signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143. Functional assays (A) NFAT-Luc and (B) SRE-Luc were conducted as for Fig. 2 at extracellular Ca2+ concentrations of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143. *P < 0.05; **P < 0.01; ***P < 0.001 relative to WT at the same NPS 2143 concentration. For each panel (A and B), values shown are the means of three replicates (SEM ≤45, not shown). Figure 3. View largeDownload slide Relative suppression of signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143. Functional assays (A) NFAT-Luc and (B) SRE-Luc were conducted as for Fig. 2 at extracellular Ca2+ concentrations of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143. *P < 0.05; **P < 0.01; ***P < 0.001 relative to WT at the same NPS 2143 concentration. For each panel (A and B), values shown are the means of three replicates (SEM ≤45, not shown). CaSR 544Q lacks the wild-type cation-π interaction in the cysteine-rich region The ECD of the human CaSR has recently been crystalized in the inactive and active states (7) (Fig. 4A). Both structures (PDB: 5K5T, inactive, and 5K5S, active) were analyzed with UCSF Chimera. A zone of 10Å radius centering on R544 was examined with respect to interactions of the arginine and its neighboring amino acids and any distance changes occurring during activation. R544 forms a cation-π interaction with F539 within the cysteine-rich domain that follows after the VFT domain (Fig. 4B). The rich electron-filled orbitals (π) of the phenylalanine aromatic ring interact with the positive charge of the arginine. In the inactive state, the interaction distance is 3.6Å and in the active state is 5.4Å, suggesting a weakening of the interaction upon ligand activation. Moreover, the cation-π interaction energy (force) predictions using the CAPTURE program are van der Waals −3.21 and −1.73 kcal/mol and electrostatic −5.02 and −1.71 kcal/mol in the inactive vs active state, respectively. This is fully consistent with the R544 and F539 cation-π interaction playing an important role in restraining the receptor in a stable inactive conformation in the unliganded state (Fig. 4C). Substitution of R544 with glutamine breaks the cation-π interaction, causing the receptor to be more responsive upon ligand activation. Figure 4. View largeDownload slide The cation-π interaction (R544-F539) in the cysteine-rich domain of the CaSR is lost when the arginine at 544 is substituted by glutamine. (A) Model of ligand activation of the CaSR dimer at the cell surface. [A(i)] Intermolecular disulfide bonds (S) link lobes (LB) 1 of the Venus fly trap (VFT) domain of each protomer (monomer) and in the absence of any ligand maintain the VFT formed by LB1 and LB2 in an open (inactive) conformation. [A(ii)] Ligand binding brings about VFT closure, a rotation about the dimer interface and causes an extended homodimer interface to form, involving not only LB1 but also LB2 and the cysteine-rich domain (Cys-rich) of each protomer. This is predicted to bring about reconfiguration of some of the transmembrane α-helices so that intracellular loops and part of the C-tail can productively contact G proteins, triggering cell signaling. (B) Active-state structure of the CaSR ECD homodimer. The positions of residues R544 and F539 are indicated in stick format and boxed. [B(i), inset) Relationship of R544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −3.21 kcal/mol; electrostatic force, −5.02 kcal/mol. [B(ii), inset] Relationship of R544 and F539 in the active state; cation-π distance, 5.4 Å; van der Waals force, −1.73 kcal/mol; electrostatic force, −1.71 kcal/mol. [B(iii), inset) Relationship of Q544 and F539 in the inactive state. The cation-π interaction is lost. [B(iv), inset] Relationship of Q544 and F539 in the active state. The cation-π interaction is lost. (C) Structural relationships of additional CaSR mutants (Mut) that were made and tested in functional assays. The positions of residues 544 and 539 are indicated in stick format and boxed. Note that for [C(a)–C(c)], insets (i) and (ii) relationships of R544 and F539 are repeated (from panel B) for comparative purposes. [C(a)(iii), inset] Relationship of K544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −1.17 kcal/mol; electrostatic force, −5.59 kcal/mol. [C(a)(iv), inset] Relationship of K544 and F539 in the active state; cation-π distance, 5.3 Å; van der Waals force, −0.54 kcal/mol; electrostatic force, −3.12 kcal/mol. [C(b)(iii), inset] Relationship of R544 and L539 in the inactive state. The cation-π interaction is lost. [C(b)(iv), inset] Relationship of R544 and L539 in the active state. The cation-π interaction is lost. [C(c)(iii), inset] Relationship of R544 and A539 in the inactive state. The cation-π interaction is lost. [C(c)(iv), inset] Relationship of R544 and A539 in the active state. The cation-π interaction is lost. Inactive- and active-state structures of the CaSR ECD homodimer, RCSB Protein Data Bank accession numbers 5K5T and 5K5S, respectively. Cation-π interaction energy predictions are from the CAPTURE program (capture.caltech.edu). Figure 4. View largeDownload slide The cation-π interaction (R544-F539) in the cysteine-rich domain of the CaSR is lost when the arginine at 544 is substituted by glutamine. (A) Model of ligand activation of the CaSR dimer at the cell surface. [A(i)] Intermolecular disulfide bonds (S) link lobes (LB) 1 of the Venus fly trap (VFT) domain of each protomer (monomer) and in the absence of any ligand maintain the VFT formed by LB1 and LB2 in an open (inactive) conformation. [A(ii)] Ligand binding brings about VFT closure, a rotation about the dimer interface and causes an extended homodimer interface to form, involving not only LB1 but also LB2 and the cysteine-rich domain (Cys-rich) of each protomer. This is predicted to bring about reconfiguration of some of the transmembrane α-helices so that intracellular loops and part of the C-tail can productively contact G proteins, triggering cell signaling. (B) Active-state structure of the CaSR ECD homodimer. The positions of residues R544 and F539 are indicated in stick format and boxed. [B(i), inset) Relationship of R544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −3.21 kcal/mol; electrostatic force, −5.02 kcal/mol. [B(ii), inset] Relationship of R544 and F539 in the active state; cation-π distance, 5.4 Å; van der Waals force, −1.73 kcal/mol; electrostatic force, −1.71 kcal/mol. [B(iii), inset) Relationship of Q544 and F539 in the inactive state. The cation-π interaction is lost. [B(iv), inset] Relationship of Q544 and F539 in the active state. The cation-π interaction is lost. (C) Structural relationships of additional CaSR mutants (Mut) that were made and tested in functional assays. The positions of residues 544 and 539 are indicated in stick format and boxed. Note that for [C(a)–C(c)], insets (i) and (ii) relationships of R544 and F539 are repeated (from panel B) for comparative purposes. [C(a)(iii), inset] Relationship of K544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −1.17 kcal/mol; electrostatic force, −5.59 kcal/mol. [C(a)(iv), inset] Relationship of K544 and F539 in the active state; cation-π distance, 5.3 Å; van der Waals force, −0.54 kcal/mol; electrostatic force, −3.12 kcal/mol. [C(b)(iii), inset] Relationship of R544 and L539 in the inactive state. The cation-π interaction is lost. [C(b)(iv), inset] Relationship of R544 and L539 in the active state. The cation-π interaction is lost. [C(c)(iii), inset] Relationship of R544 and A539 in the inactive state. The cation-π interaction is lost. [C(c)(iv), inset] Relationship of R544 and A539 in the active state. The cation-π interaction is lost. Inactive- and active-state structures of the CaSR ECD homodimer, RCSB Protein Data Bank accession numbers 5K5T and 5K5S, respectively. Cation-π interaction energy predictions are from the CAPTURE program (capture.caltech.edu). Discussion This report describes an unusual case in which the proband presented as ADH1 but was homozygous for a CASR variant. Classically, ADH1 cases are hypocalcemic (mild to moderate) with hyperphosphatemia, relative hypercalciuria, and inappropriately low but detectable PTH levels (2). However, not all ADH1-affected individuals exhibit relative or absolute hypercalciuria (and nephrocalcinosis) (26–28), and this was the case for the proband of the family who was studied here. The proband had basal ganglia calcification and cataracts. Basal ganglia calcification is common in patients with ADH1 (25–30). A mouse strain (designated “Nuf”) harboring an activating CaSR mutation (L723Q) developed ectopic calcification and cataracts (31). The plasma calcium levels in the homozygous Nuf/Nuf mice were more significantly reduced than those in the heterozygous Nuf/+ mice, which were significantly reduced from those in the wild-type mice. Hyperphosphatemia was significantly greater in both the heterozygous and homozygous mice, with a clear trend to higher levels in the homozygous mice than in the heterozygous mice. Also, the heterozygous and homozygous mice were not hypercalciuric (31). In fact, the female Nuf/Nuf mice were hypocalciuric, similar to the proband of the present case. In addition, the ectopic calcification and cataracts were milder in the heterozygous mice than in the homozygous mice (31). Careful evaluation for cataracts should be done in ADH1 cases. With respect to mechanism, the following was suggested (31). The CaSR is expressed in the lens epithelial cells (32), and the elevated intracellular Ca2+ associated with cataract formation (33) triggers the activation of calpain, which modifies cytoskeletal proteins and β-crystallin in lens cataract models (33, 34). Hence, the overactive CaSR mutant may trigger the increases in intracellular Ca2+ that lead to cataract development. CASR R544Q is represented 340 times (as the heterozygote) in public single nucleotide polymorphism databases, with a frequency approaching 0.1%. In the ClinVar database that reports on the relationships among human variations and phenotypes, the eight entries (cases) designated as R544Q cover the spectrum from hypocalcemic to hypercalcemic disorders. In addition, in a single published report, the R544Q variant was found in an individual with familial hypocalciuric hypercalcemia (24). In silico testing assigns the variant as benign (PolyPhen) or tolerated (SIFT). The CaSR couples to intracellular signaling pathways via Gq/11, which leads to increases in intracellular Ca2+, and Gi, which leads to activation of MAPK family members. Our in vitro functional assays examined both of these pathways. In each type of assay, R544Q alone, mimicking the situation in the proband of the present family, was significantly more active than WT in the extracellular Ca2+ dose-response curve. However, R544Q was not more significantly active than WT when coexpressed with WT to mimic the heterozygous state in the parents of the proband. This is fully consistent with the phenotypic expression of R544Q as the homozygote but not as the heterozygote. For comparative purposes, we found that the previously described ADH1 CaSR E604K mutant was significantly more active than the WT alone when evaluated alone and when coexpressed with WT. Moreover, studies with the calcilytic NPS 2143 emphasized the mild overactivity of R544Q relative to the marked overactivity of the E604K mutation. The determination of the crystallographic structure of the CaSR ECD (7) highlighted the important role of the cysteine-rich domain in coupling ligand-induced closure of the VFT domain to the proposed altered conformation of the TMD and interaction with G proteins and signal pathway activation. The cation-π interaction is a stabilizing electrostatic interaction of a cation with the polarizable π electron cloud of an aromatic ring. The cation-π interaction is held to be as important as the hydrophobic effect, the hydrogen bond, or the ion pair in determining macromolecular structure (17, 35). Cation-π interactions have been documented in many proteins in which the arginine or lysine side chains interact with phenylalanine, tyrosine, or tryptophan. The CaSR ECD has five cation-π interactions, and one of these is R544-F539 within the cysteine-rich domain. In the ligand-activated CaSR, the distance between R544 and F539 increases and the electrostatic forces between them weaken. This is consistent with the cation-π interaction contributing to maintenance of the receptor in an inactive conformation when the ligand is absent. Ligand activation overcomes this restraining force, but the cation-π interaction appears to set the potency of the activation, because when it is lost in the R544Q mutant, the potency of activation of the receptor is increased. In our study, we provide further evidence of the nature and importance of the R544-F539 cation-π interaction by the finding that the engineered R544K mutant, which preserves the cationic residue, behaves identically to WT in functional assays, whereas mutant F539A and variant F539L that have lost the aromatic π orbitals are overactive, similar to R544Q. In conclusion, we report on the identification of a rare missense variant in the CaSR cysteine-rich domain that, while not clinically expressed as the heterozygote, came to attention in an individual homozygous for the variant. This has important implications for diagnosis, clinical management, and genetic counseling. Our study also revealed a particular structural feature of the CaSR––a cation-π interaction––that, when lost, makes the receptor more responsive to extracellular calcium. Abbreviations: Abbreviations: ADH1 autosomal dominant hypocalcemia type 1 [Ca2+]o extracellular Ca2+ concentration CASR calcium-sensing receptor gene ECD extracellular domain ExAC Exome Aggregation Consortium GCM2 glial cells missing-2 gene GNA11 G protein α11 gene HEK human embryonic kidney Luc luciferase NFAT nuclear factor of activated T cells NR normal range NS not significant SRE serum response element TMD transmembrane domain VFT Venus flytrap‒like WT wild type Acknowledgments We thank the family members for their participation. We thank NPS Pharmaceuticals, Inc., Salt Lake City, Utah, and Dr. Jenny Yang for HEK293 cells; Drs. Tajima and Fukumoto for promoter-reporter constructs; and Dr. Svetlana Pidasheva and Sarah Elliott for assistance. The authors are also grateful to Nicole Fabien (Service d’Immunologie, UF Autoimunité, Centre Hospitalier, Lyon Sud, France) for the assessment of CaSR antibodies in the proband, to Sara Donato for providing family members’ clinical data, and to Luís Sobrinho for support in the clinical/metabolic interpretation of the case. Financial Support: iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia/Ministério da Ciência e do Ensino Superior, through national funds, Associação de Endocrinologia Oncológica, Lisboa, Portugal, and FEDER under the PT2020 Partnership Agreement are acknowledged. This study was supported by a Canadian Institutes of Health Research operating grant (to G.N.H.). Disclosure Summary: The authors have nothing to disclose. References 1. Hendy GN , Cole DEC . Familial isolated hypoparathyroidism. In: Brandi ML , Brown EM , eds. Hypoparathyroidism . Milan, Italy: Springer-Verlag Italia ; 2015 : 167 – 175 . 2. 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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-02407
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Abstract

Abstract Context Autosomal dominant hypocalcemia type 1 (ADH1) is caused by heterozygous activating mutations in the calcium-sensing receptor gene (CASR). Whether polymorphisms that are benign in the heterozygous state pathologically alter receptor function in the homozygous state is unknown. Objective To identify the genetic defect in an adolescent female with a history of surgery for bilateral cataracts and seizures. The patient has hypocalcemia, hyperphosphatemia, and low serum PTH level. The parents of the proband are healthy. Methods Mutation testing of PTH, GNA11, GCM2, and CASR was done on leukocyte DNA of the proband. Functional analysis in transfected cells was conducted on the gene variant identified. Public single nucleotide polymorphism (SNP) databases were searched for the presence of the variant allele. Results No mutations were identified in PTH, GNA11, and GCM2 in the proband. However, a germline homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR was identified. Both parents are heterozygous for the variant. The variant allele frequency was near 0.1% in SNP databases. By in vitro functional analysis, the variant was significantly more potent in stimulating both the Ca2+i and MAPK signaling pathways than wild type when transfected alone (P < 0.05) but not when transfected together with wild type. The overactivity of the mutant CaSR is due to loss of a critical structural cation-π interaction. Conclusions The patient’s hypoparathyroidism is due to homozygosity of a variant in the CASR that normally has weak or no phenotypic expression in heterozygosity. Although rare, this has important implications for genetic counseling and clinical management. Congenital or familial isolated hypoparathyroidism may present in an autosomal dominant or recessive fashion and can be due to mutations in the calcium-sensing receptor gene (CASR), glial cells missing-2 gene (GCM2), G protein α11 gene (GNA11), or parathyroid hormone gene (PTH) (1). Gain-of-function mutations in the CASR (MIM#601199) have been identified in a number of families with autosomal dominant hypocalcemia type 1 (ADH1) (MIM#601198). In the parathyroid gland, the activated CaSR (the calciostat) suppresses PTH secretion, and in the kidney, in some cases it induces hypercalciuria that may contribute to the hypocalcemia (2, 3). The CaSR is a member of G protein‒coupled receptor family C. The mature human protein has an extracellular domain (ECD) of 593 amino acids, a 250‒amino acid transmembrane domain (TMD), and a 216‒amino acid intracellular tail (4, 5). The CaSR functions as a dimer. The ECD of each CaSR monomer has a bilobed Venus flytrap‒like (VFT) domain followed by a cysteine-rich domain and a linker region before the TMD (5, 6). Recently, the crystal structure of the ECD of the CaSR was elucidated to provide insights into binding to the VFT of Ca2+ and Mg2+ and amino acids and subsequent conformational changes leading to activation (7, 8). Of the ADH1 mutations identified to date, >65 are located in the ECD, and three-quarters of these are found at the dimer interface, with the remainder at the ligand-binding or other sites (3). In a study in which the structure of the entire ECD was determined, the importance of the cysteine-rich domain in contributing to the extended dimerization interface upon ligand binding was revealed (7). Several signaling pathways are activated by the CaSR (9). The stimulated CaSR couples to Gq/11, causing phospholipase C‒mediated production of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the latter of which causes intracellular Ca2+ mobilization from endoplasmic reticulum stores. It also activates MAPK family members (9). In some cases, in vitro functional testing of mutant CaSRs has been critical in correlating the clinical presentation with the relative overactivity of particular ADH1 mutations (10, 11). In the current study, we report the identification of a rare missense variant in the CaSR cysteine-rich domain that, while not clinically expressed in the heterozygous state, came to our attention in an individual who is homozygous for the variant. Our study also revealed a particular structural feature of the CaSR—a cation-π interaction—that when lost makes the receptor more responsive to extracellular calcium. Patient and Methods Case report The proband is a 20-year-old female patient with hypoparathyroidism (II-1; Fig. 1; Table 1). She was born a healthy baby at 40 weeks (weight, 3800 g; length, 52 cm), and her perinatal history was uneventful. Her psychomotor development was normal, with no evidence of tetany or seizures. She was breastfed until 2.5 months of age and was supplemented with 25-hydroxyvitamin D until the age of 1 year. At age 16 years, she underwent surgery for bilateral cataracts. She was evaluated at 18 years of age for an episode of seizures without any intercurrent illness. Laboratory analysis showed hypocalcemia, 4.4 mg/dL [normal range (NR) = 8.4 to 10.2 mg/dL], hyperphosphatemia, 8.7 mg/dL (NR = 2.3 to 4.7 mg/dL), undetectable serum PTH level, <2.5 pg/mL (NR = 12 to 65 pg/mL), and hypocalciuria, 0.02 g/L (NR = 0.05 to 1.15 g/L) (Table 1). At her first visit to our clinic at 20 years of age, she was under calcium (1200 mg/d) and calcitriol (0.5 µg/d) treatment. Her serum calcium level was 6.6 mg/dL, with hyperphosphatemia (8.2 mg/dL) and normal levels of 25-hydroxyvitamin D (32 ng/mL; NR = 9 to 45 ng/mL) (Table 1). She did not demonstrate paresthesia, cramps, or tetany, and Chvostek and Trousseau signs were negative. Electrocardiography revealed a prolonged QTc interval (451 ms). Brain CT showed basal ganglia calcification. Nephrocalcinosis was excluded by renal ultrasonography. Polyglandular autoimmune syndrome was excluded on laboratory and clinical grounds, and patient serum was negative for CaSR antibodies. Figure 1. View largeDownload slide Detection of a variant in the CASR gene. (A) Pedigree of family with ADH1 and CASR variant: open symbol, unaffected; filled symbol, affected. Proband is indicated by the arrow. CASR genotypes; +, wild-type allele; R554Q, variant allele. (B) Direct sequence analysis of CASR exon 6 genomic DNA amplicon revealed heterozygosity in parents I-1 and I-2 and homozygosity in proband II-1 for the c.1631G>A; p.R544Q variant. (C) The CaSR protein sequences from diverse species were aligned as described in Materials and Methods. The residue corresponding to the R544Q variant is boxed as are the conserved C542 and C546 residues in the CaSR cysteine-rich domain. (D) Western blot analysis of extracts of HEK293 cells transiently transfected with vector (pcDNA3.1), wild type (WT), or variant (R544Q) c-Myc‒tagged CaSR cDNAs. (Di) Recombinant proteins stained with c-Myc monoclonal antibody 9E10. (Dii) Endogenous proteins stained with a tubulin antibody. Bo, bovine; Ch, chicken; Hu, human; Mo, mouse; Ra, Rat; Ze; zebrafish. Figure 1. View largeDownload slide Detection of a variant in the CASR gene. (A) Pedigree of family with ADH1 and CASR variant: open symbol, unaffected; filled symbol, affected. Proband is indicated by the arrow. CASR genotypes; +, wild-type allele; R554Q, variant allele. (B) Direct sequence analysis of CASR exon 6 genomic DNA amplicon revealed heterozygosity in parents I-1 and I-2 and homozygosity in proband II-1 for the c.1631G>A; p.R544Q variant. (C) The CaSR protein sequences from diverse species were aligned as described in Materials and Methods. The residue corresponding to the R544Q variant is boxed as are the conserved C542 and C546 residues in the CaSR cysteine-rich domain. (D) Western blot analysis of extracts of HEK293 cells transiently transfected with vector (pcDNA3.1), wild type (WT), or variant (R544Q) c-Myc‒tagged CaSR cDNAs. (Di) Recombinant proteins stained with c-Myc monoclonal antibody 9E10. (Dii) Endogenous proteins stained with a tubulin antibody. Bo, bovine; Ch, chicken; Hu, human; Mo, mouse; Ra, Rat; Ze; zebrafish. Table 1. Biochemical Characteristics of Family Members Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Abbreviation: 25(OH)D, 25-hydroxyvitamin D. a Normal ranges in parentheses. b Before treatment with calcium and calcitriol. c On treatment with calcium and calcitriol. View Large Table 1. Biochemical Characteristics of Family Members Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Pedigree Serum Calcium (8.4–10.2 mg/dL)a Serum Phosphate (2.3–4.7 mg/dL) Serum Creatinine (0.6–1.2 mg/dL) Serum Magnesium (1.6–2.6 mg/dL) Serum PTH (12–65 pg/mL) Serum 25(OH)D (9–45 ng/mL) Urinary calcium (100–300 mg/24 h) I–1 9.1 2.5 0.9 2.0 17.0 15.0 118 I–2 9.9 3.7 0.7 2.0 20.7 32.0 214 II–1 4.4b (6.6c) 8.7b (8.2c) 0.9b (0.7c) 1.8b <2.5b,c 32.0c 77c Abbreviation: 25(OH)D, 25-hydroxyvitamin D. a Normal ranges in parentheses. b Before treatment with calcium and calcitriol. c On treatment with calcium and calcitriol. View Large Both parents (individuals I-1 and I-2; Fig. 1; Table 1) are clinically healthy with normal serum calcium and phosphate levels and normal fractional excretion of calcium. The parents are unaware of consanguinity, although they do share identical haplotypes at the CASR locus (Supplemental Table 1), which suggests they may have a common ancestor. DNA sequence analysis Written informed consent was obtained for blood collection and analyses of relevant genes in the proband and her parents. In brief, the analysis of the CASR, GNA11, PTH, and GCM2 genes involved genomic DNA extraction from peripheral blood leukocytes, PCR amplification of the gene exons including the adjoining splice junctions (primer sequences are available on request), and direct sequencing of the PCR products. Publicly accessible databases were examined for the presence of the identified sequence variant: the Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org/), representing exomes of 60,706 unrelated individuals (12); the Genome Aggregation Database (gnomad.broadinstitute.org/), representing 277,238 alleles (from ExAc and other sources); the ClinVar (www.ncbi.nlm.nih.gov/clinvar/), an archive of reports of the relationships among human variations and phenotypes (13); and the Human Gene Mutation Database (www.hgmd.cf.ac.uk/ac/index.php/), which collates known (published) gene lesions responsible for human inherited disease. Protein sequence alignment and three-dimensional modeling of CaSR structure Protein sequences of CaSR orthologues were aligned using ClustalOmega (www.ebi.ac.uk/Tools/msa/clustalo/) (14). The crystal structures of the human CaSR ECD in the inactive (5K5T) and active (5K5S) conformations (7) were obtained from the Protein Data Bank at www.rcsb.org (15) and analyzed using UCSF Chimera (16). The mutation tool that replaces one amino acid with another while selecting the rotamer with the highest χ2 statistic without directly affecting neighboring amino acids was used to replace R544 with Q544. Interactions between R544 and other amino acids within a 10Å radius were evaluated for inactive and active states of the receptor. Cation-π interactions and their energies were predicted with the CAPTURE program (17). The PyMOL Molecular Graphics System (PyMOL v1.8.6.2) (www.pymol.org/) was used to visualize protein structure. Site-directed mutagenesis and transfection Mutations were introduced into a c-Myc‒tagged human CaSR cDNA in pcDNA3.1 (18). The correctness of the constructs was confirmed by sequencing. Human embryonic kidney (HEK) 293 cells were transfected with c-Myc‒tagged human CaSR cDNAs, WT or mutant (19). Western blot analysis Proteins were isolated from HEK293 cells transiently transfected with WT or mutant c-Myc‒tagged CaSR cDNAs and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis through a 6% to 10% linear gradient gel, followed by Western blotting (19). Functional analysis of CaSRs Increased intracellular Ca2+ activates the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway. An NFAT promoter‒luciferase (Luc) assay was used to analyze the responsiveness of each mutant CaSR to extracellular Ca2+ (11). HEK293 cells were plated at 1 × 106 cells per well in six-well dishes in DMEM (#D9785; Sigma-Aldrich, Oakville, ON, Canada) plus 10% fetal bovine serum and cultured overnight to 70% confluency. Cells were then transiently cotransfected with 1.0 μg of WT or mutant CaSR constructs or empty vector or with 0.5 μg of WT and 0.5 μg of mutant CaSR, 0.5 μg of pGL3-NFAT-Luc reporter vector (plasmid 17870; Addgene, Cambridge, MA), and 0.5 μg of pRSV-Gal β-galactosidase vector using 20 μL per well PolyFect Transfection Reagent (Qiagen, Valencia, CA). After 16 hours, cells were serum starved in DMEM containing 0.5 mM of CaCl2, 0.5 mM of MgCl2 for 8 hours and treated with CaCl2 concentrations from 0.5 to 20 mM for 16 hours. Cells were then washed twice with PBS and lysed in 100 μL per well passive lysis buffer (Promega, Madison, WI). Luc activity was measured in a FLUOstar Optima Luminometer (BMG Labtech, Guelph, ON, Canada) and expressed relative to β-galactosidase activity. The activation of the ERK/MAPK pathway by the CaSR constructs in response to extracellular Ca2+ was made with pGL4.33 (Promega), a serum response element (SRE) promoter‒Luc reporter vector (10, 20). HEK293 cells were plated in six-well plates and grown to 70% confluency and transiently transfected using 20 μL per well PolyFect Transfection Reagent (Qiagen) with 1.0 μg of WT or mutant CaSR or empty vector or with 0.5 μg of WT and 0.5 μg of mutant CaSR, 0.5 μg of pGL4.33, and 0.5 μg of pRSV-Gal β-galactosidase vector. The following day, cells were serum-starved in DMEM containing 0.5 mM CaCl2, 0.5 mM MgCl2 for 8 hours and cultured in various concentrations of CaCl2 ranging from 0.5 to 20 mM for 16 hours. Cell extracts were prepared, and relative Luc activity was determined. A heterozygous CASR c.1810G>A; p.E604K mutation has been described in affected members of three independent families with ADH1 (21–23). In vitro functional studies have documented that CaSR E604K is properly expressed at the plasma membrane of transfected cells and is more active than WT CaSR in extracellular Ca2+ signaling pathway assays (21). We prepared a CaSR E604K expression construct and used it in functional assays as a reference for the relative activities obtained with the CaSR R544Q construct. Effect of negative allosteric modulator (NPS 2143) Functional assays were conducted as described previously, with 3.0 or 5.0 mM of Ca2+ as these concentrations bracket the EC50 value for the dose-response curve of the wild-type CaSR. At the time of changing the cell media to these Ca2+ concentrations, 50, 100, or 500 nM of NPS 2143 (synthesized in-house) in 0.1% dimethyl sulfoxide (or vehicle only) was added, and the incubation continued for 16 hours before harvest, cell lysis, and Luc assay. Statistical analysis Data are mean ± SEM. Results shown are representative of assays repeated three times. Nonlinear regression of concentration-response curves was performed with GraphPad Prism (La Jolla, CA) using the normalized response at each extracellular Ca2+ concentration [(Ca2+)o] for the determination of EC50 [i.e., (Ca2+)o required for 50% of the maximal response]. Statistical comparisons were made with the Mann-Whitney U test. A P value < 0.05 was considered significant. Supplemental methods CaSR intact cell surface ELISA, crude plasma membrane preparation and western blotting, and IP-One Assay (Cisbio, Burlington, ON, Canada) are described in online Supplemental Data. Results Identification of CASR variant No mutations were identified in the PTH, GNA11, and GCM2 genes in the germline DNA of the proband (II-1; Fig. 1A). However, a homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR gene was identified (Fig. 1B). Both parents (I-1 and I-2; Fig. 1A) were heterozygous for the variant (Fig. 1B). An arginine at position 544 is highly conserved among species (Fig. 1C). The R544Q variant does appear in single nucleotide polymorphism databases at a frequency of 0.08% (ExAC) and 0.09% (Genome Aggregation Database) (Table 2). The variant appears in the ClinVar database under eight entries, with descriptions covering both hypercalcemic (hyperparathyroid) and hypocalcemic (hypoparathyroid) conditions (Table 2). The clinical significance of these entries is given as likely benign or uncertain. A published report documents heterozygosity of the R544Q variant in an individual with familial hypocalciuric hypercalcemia (24). All entries in databases and reports are for R544Q as a heterozygote. Table 2. R544Q Variants in Public Databasesa Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Abbreviations: FHH1, familial hypocalciuric hypercalcemia type 1; gnomAD, Genome Aggregation Database. a All variants are heterozygotes. b Single entries of c.1631G>T; R544L and c.1630C>T; R544Ter occur in the databases. c In ExAc and gnomAD databases, R554Q is identified (rather than R544Q) as a different splice variant of the CaSR mRNA is used as the reference that encodes 10 extra amino acids (1088 rather than 1078), increasing the number by 10 after amino acid 536. Major form: NM_000388.3, NP_000379.2, 1078 amino acids; minor form: NM_001178065.1, NP_001171536.1, 1088 amino acids. View Large Table 2. R544Q Variants in Public Databasesa Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Database c.1631G>A; p.R544Qb ExAcc Allele count: 96 European (Non-Finnish): 89 Latino: 4; African: 3 Allele frequency: 96/121410 (0.0007907) gnomADc Allele count: 244 Azkenazi Jewish: 62 European (Non-Finnish): 157 Other: 4; Latino: 12; African: 6 Allele frequency: 244/277238 (0.0008801) ClinVar Eight entries: Conditions (mode of inheritance) of each case described as follows: (1) Hypocalciuric hypercalcemia familial type 1 (2) Hypocalcemia autosomal dominant type 1 (3) Not specified (4) Hypocalcemia (5) Familial hypocalciuric hypercalcemia (6) Neonatal severe hyperparathyroidism (7) Hypoparathyroidism familial isolated (8) Not specified Clinical significance of R544Q in these cases ascribed as follows: five cases, likely benign; three cases, uncertain significance HGMD One entry: FHH1 patient (24) Abbreviations: FHH1, familial hypocalciuric hypercalcemia type 1; gnomAD, Genome Aggregation Database. a All variants are heterozygotes. b Single entries of c.1631G>T; R544L and c.1630C>T; R544Ter occur in the databases. c In ExAc and gnomAD databases, R554Q is identified (rather than R544Q) as a different splice variant of the CaSR mRNA is used as the reference that encodes 10 extra amino acids (1088 rather than 1078), increasing the number by 10 after amino acid 536. Major form: NM_000388.3, NP_000379.2, 1078 amino acids; minor form: NM_001178065.1, NP_001171536.1, 1088 amino acids. View Large Expression of wild-type and variant CaSRs in HEK293 cells The CaSR 544Q variant was expressed at equivalent levels and exhibited the same pattern of molecular species as CaSR WT: the core glycosylated (immature) 140-kDa species and the mature, fully glycosylated 160-kDa species (Fig. 1D). High molecular mass forms (∼300 kDa), likely to be dimers, were seen equally for the variant and WT receptors (Fig. 1D). The data show the variant achieved proper maturation and was appropriately trafficked to the plasma membrane under a variety of conditions (Supplemental Figs. 1 and 2). Responsiveness of the CaSR R544Q variant to extracellular calcium The ability of the variant receptor to respond to extracellular calcium relative to the WT receptor was assessed in three different assays. In the NFAT-Luc assay that monitors changes in intracellular Ca2+, the WT CaSR showed an EC50 of 4.3 ± 0.16 mM Ca2+ (Fig. 2A). The variant R544Q showed a significant (P < 0.05) leftward shift in its concentration-response curve (EC50, 3.6 ± 0.23 mM) relative to that of WT. When equal amounts of WT and variant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.95 ± 0.19 mM) to a position intermediate to, but not significantly different from, that of WT alone [not significant (NS)] and the variant alone (NS). Figure 2. View largeDownload slide (A) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either wild-type alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the NFAT-Luc promoter reporter construct. (B) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the SRE-Luc promoter reporter construct. (C) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), E604K and WT together (E604K/WT), or E604K alone (E604K) and the SRE-Luc promoter reporter construct. (D) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the NFAT-Luc promoter reporter construct. (E) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the SRE-Luc promoter reporter construct. (F) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or F539A and the NFAT-Luc promoter reporter construct. For each panel (A–F), values shown are the means of three replicates (SEM ≤45, not shown). Figure 2. View largeDownload slide (A) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either wild-type alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the NFAT-Luc promoter reporter construct. (B) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), R544Q and WT together (R544Q/WT), or R544Q alone (R544Q) and the SRE-Luc promoter reporter construct. (C) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT alone (WT), E604K and WT together (E604K/WT), or E604K alone (E604K) and the SRE-Luc promoter reporter construct. (D) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the NFAT-Luc promoter reporter construct. (E) Extracellular calcium-evoked increases in MAPK activity in HEK293 cells transiently transfected with either WT, R544Q, or R544K and the SRE-Luc promoter reporter construct. (F) Extracellular calcium-evoked increases in intracellular Ca2+ in HEK293 cells transiently transfected with either WT, R544Q, or F539A and the NFAT-Luc promoter reporter construct. For each panel (A–F), values shown are the means of three replicates (SEM ≤45, not shown). In the SRE-Luc assay that monitors changes in ERK/MAPK activity, the WT CaSR cDNA showed an EC50 of 4.1 ± 0.16 mM Ca2+ (Fig. 2B). The variant R544Q showed a significant (P < 0.05) leftward shift in its concentration-response curve (EC50, 3.3 ± 0.18 mM) relative to that of WT. When equal amounts of WT and variant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.7 ± 0.19 mM) to a position intermediate to, but not significantly different from, that of WT alone (NS) and the variant alone (NS). Hence, in both types of promoter-Luc assay, the variant alone had a significantly greater potency than WT CaSR alone but not when cotransfected with WT CaSR. The ADH1 CaSR E604K mutant was used as a reference control in the SRE-Luc assay. The WT CaSR showed an EC50 of 4.1 ± 0.16 mM Ca2+ (Fig. 2C). The mutant E604K showed a significant (P < 0.01) leftward shift in its concentration-response curve (EC50, 3.1 ± 0.13 mM) relative to that of WT. When equal amounts of WT and mutant CaSR cDNAs were transiently coexpressed, the concentration-response curve was leftward shifted (EC50, 3.6 ± 0.14 mM) to a position intermediate to, but significantly different from, that of WT alone (P < 0.05) and the mutant alone (P < 0.05). Similar findings were obtained in the NFAT-Luc assay (Supplemental Fig. 3). Hence, the E604K mutant alone had a significantly greater potency than WT CaSR alone and also when cotransfected with WT CaSR in contrast to CaSR R544Q (Fig. 2A and 2B). In the IP-One assay that monitors changes in inositol phosphate, similar findings were obtained for the relative potencies of WT alone, R544Q/WT together, and R544Q alone (Supplemental Fig. 4A) and WT alone, E604K/WT together, and E604K alone (Supplemental Fig. 4), as in the NFAT-Luc and SRE-Luc assays. Further engineered mutants were evaluated as follows. An R544K mutant demonstrated potency identical to that of WT in both NFAT-Luc (Fig. 2D) and SRE-Luc (Fig. 2E) assays. An F539A mutant demonstrated enhanced potency similar to that of the R544Q variant in both NFAT-Luc (Fig. 2F) and SRE-Luc (Supplemental Fig. 5A) assays. The F539L variant demonstrated enhanced potency similar to that of the R544Q variant in both NFAT-Luc (Supplemental Fig. 5B) and SRE-Luc (Supplemental Fig. 5C) assays. A lower dose of NPS 2143 is required to restore the activity of R544Q to that of WT CaSR relative to an ADH1 CaSR mutant The calcilytic NPS 2143 has the ability to decrease the responsiveness of overactive mutant CaSRs to extracellular Ca2+ (10, 25). The relative amount of NPS 2143 required to restore the activity of a particular ADH1 CaSR mutant to that of WT CaSR indicates its relative potency as a gain-of-function mutant. Relative suppression of the signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143 was evaluated. A functional NFAT-Luc assay was conducted at [Ca2+]o of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143 (Fig. 3). Although 50 nM NPS 2143 was sufficient to restore the activity of CaSR R544Q to that of CaSR WT in the absence of the calcilytic at both [Ca2+]o of 3 and 5 mM, CaSR E604K remained highly significantly overactive at this particular NPS 2143 concentration (Fig. 3A). Increasing concentrations (100 and 500 nM) of NPS 2143 reduced the activities of both R544Q and E604K further while maintaining the relationship of much less activity for the R544Q variant relative to the E604K mutant at each particular NPS 2143 concentration tested (Fig. 3A). Similar findings were obtained in SRE-Luc assays (Fig. 3B). Figure 3. View largeDownload slide Relative suppression of signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143. Functional assays (A) NFAT-Luc and (B) SRE-Luc were conducted as for Fig. 2 at extracellular Ca2+ concentrations of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143. *P < 0.05; **P < 0.01; ***P < 0.001 relative to WT at the same NPS 2143 concentration. For each panel (A and B), values shown are the means of three replicates (SEM ≤45, not shown). Figure 3. View largeDownload slide Relative suppression of signaling activity of CaSR WT, R544Q, and E604K by the calcilytic NPS 2143. Functional assays (A) NFAT-Luc and (B) SRE-Luc were conducted as for Fig. 2 at extracellular Ca2+ concentrations of 3 or 5 mM and in the absence or presence of 50, 100, or 500 nM NPS 2143. *P < 0.05; **P < 0.01; ***P < 0.001 relative to WT at the same NPS 2143 concentration. For each panel (A and B), values shown are the means of three replicates (SEM ≤45, not shown). CaSR 544Q lacks the wild-type cation-π interaction in the cysteine-rich region The ECD of the human CaSR has recently been crystalized in the inactive and active states (7) (Fig. 4A). Both structures (PDB: 5K5T, inactive, and 5K5S, active) were analyzed with UCSF Chimera. A zone of 10Å radius centering on R544 was examined with respect to interactions of the arginine and its neighboring amino acids and any distance changes occurring during activation. R544 forms a cation-π interaction with F539 within the cysteine-rich domain that follows after the VFT domain (Fig. 4B). The rich electron-filled orbitals (π) of the phenylalanine aromatic ring interact with the positive charge of the arginine. In the inactive state, the interaction distance is 3.6Å and in the active state is 5.4Å, suggesting a weakening of the interaction upon ligand activation. Moreover, the cation-π interaction energy (force) predictions using the CAPTURE program are van der Waals −3.21 and −1.73 kcal/mol and electrostatic −5.02 and −1.71 kcal/mol in the inactive vs active state, respectively. This is fully consistent with the R544 and F539 cation-π interaction playing an important role in restraining the receptor in a stable inactive conformation in the unliganded state (Fig. 4C). Substitution of R544 with glutamine breaks the cation-π interaction, causing the receptor to be more responsive upon ligand activation. Figure 4. View largeDownload slide The cation-π interaction (R544-F539) in the cysteine-rich domain of the CaSR is lost when the arginine at 544 is substituted by glutamine. (A) Model of ligand activation of the CaSR dimer at the cell surface. [A(i)] Intermolecular disulfide bonds (S) link lobes (LB) 1 of the Venus fly trap (VFT) domain of each protomer (monomer) and in the absence of any ligand maintain the VFT formed by LB1 and LB2 in an open (inactive) conformation. [A(ii)] Ligand binding brings about VFT closure, a rotation about the dimer interface and causes an extended homodimer interface to form, involving not only LB1 but also LB2 and the cysteine-rich domain (Cys-rich) of each protomer. This is predicted to bring about reconfiguration of some of the transmembrane α-helices so that intracellular loops and part of the C-tail can productively contact G proteins, triggering cell signaling. (B) Active-state structure of the CaSR ECD homodimer. The positions of residues R544 and F539 are indicated in stick format and boxed. [B(i), inset) Relationship of R544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −3.21 kcal/mol; electrostatic force, −5.02 kcal/mol. [B(ii), inset] Relationship of R544 and F539 in the active state; cation-π distance, 5.4 Å; van der Waals force, −1.73 kcal/mol; electrostatic force, −1.71 kcal/mol. [B(iii), inset) Relationship of Q544 and F539 in the inactive state. The cation-π interaction is lost. [B(iv), inset] Relationship of Q544 and F539 in the active state. The cation-π interaction is lost. (C) Structural relationships of additional CaSR mutants (Mut) that were made and tested in functional assays. The positions of residues 544 and 539 are indicated in stick format and boxed. Note that for [C(a)–C(c)], insets (i) and (ii) relationships of R544 and F539 are repeated (from panel B) for comparative purposes. [C(a)(iii), inset] Relationship of K544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −1.17 kcal/mol; electrostatic force, −5.59 kcal/mol. [C(a)(iv), inset] Relationship of K544 and F539 in the active state; cation-π distance, 5.3 Å; van der Waals force, −0.54 kcal/mol; electrostatic force, −3.12 kcal/mol. [C(b)(iii), inset] Relationship of R544 and L539 in the inactive state. The cation-π interaction is lost. [C(b)(iv), inset] Relationship of R544 and L539 in the active state. The cation-π interaction is lost. [C(c)(iii), inset] Relationship of R544 and A539 in the inactive state. The cation-π interaction is lost. [C(c)(iv), inset] Relationship of R544 and A539 in the active state. The cation-π interaction is lost. Inactive- and active-state structures of the CaSR ECD homodimer, RCSB Protein Data Bank accession numbers 5K5T and 5K5S, respectively. Cation-π interaction energy predictions are from the CAPTURE program (capture.caltech.edu). Figure 4. View largeDownload slide The cation-π interaction (R544-F539) in the cysteine-rich domain of the CaSR is lost when the arginine at 544 is substituted by glutamine. (A) Model of ligand activation of the CaSR dimer at the cell surface. [A(i)] Intermolecular disulfide bonds (S) link lobes (LB) 1 of the Venus fly trap (VFT) domain of each protomer (monomer) and in the absence of any ligand maintain the VFT formed by LB1 and LB2 in an open (inactive) conformation. [A(ii)] Ligand binding brings about VFT closure, a rotation about the dimer interface and causes an extended homodimer interface to form, involving not only LB1 but also LB2 and the cysteine-rich domain (Cys-rich) of each protomer. This is predicted to bring about reconfiguration of some of the transmembrane α-helices so that intracellular loops and part of the C-tail can productively contact G proteins, triggering cell signaling. (B) Active-state structure of the CaSR ECD homodimer. The positions of residues R544 and F539 are indicated in stick format and boxed. [B(i), inset) Relationship of R544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −3.21 kcal/mol; electrostatic force, −5.02 kcal/mol. [B(ii), inset] Relationship of R544 and F539 in the active state; cation-π distance, 5.4 Å; van der Waals force, −1.73 kcal/mol; electrostatic force, −1.71 kcal/mol. [B(iii), inset) Relationship of Q544 and F539 in the inactive state. The cation-π interaction is lost. [B(iv), inset] Relationship of Q544 and F539 in the active state. The cation-π interaction is lost. (C) Structural relationships of additional CaSR mutants (Mut) that were made and tested in functional assays. The positions of residues 544 and 539 are indicated in stick format and boxed. Note that for [C(a)–C(c)], insets (i) and (ii) relationships of R544 and F539 are repeated (from panel B) for comparative purposes. [C(a)(iii), inset] Relationship of K544 and F539 in the inactive state; cation-π distance, 3.6 Å; van der Waals force, −1.17 kcal/mol; electrostatic force, −5.59 kcal/mol. [C(a)(iv), inset] Relationship of K544 and F539 in the active state; cation-π distance, 5.3 Å; van der Waals force, −0.54 kcal/mol; electrostatic force, −3.12 kcal/mol. [C(b)(iii), inset] Relationship of R544 and L539 in the inactive state. The cation-π interaction is lost. [C(b)(iv), inset] Relationship of R544 and L539 in the active state. The cation-π interaction is lost. [C(c)(iii), inset] Relationship of R544 and A539 in the inactive state. The cation-π interaction is lost. [C(c)(iv), inset] Relationship of R544 and A539 in the active state. The cation-π interaction is lost. Inactive- and active-state structures of the CaSR ECD homodimer, RCSB Protein Data Bank accession numbers 5K5T and 5K5S, respectively. Cation-π interaction energy predictions are from the CAPTURE program (capture.caltech.edu). Discussion This report describes an unusual case in which the proband presented as ADH1 but was homozygous for a CASR variant. Classically, ADH1 cases are hypocalcemic (mild to moderate) with hyperphosphatemia, relative hypercalciuria, and inappropriately low but detectable PTH levels (2). However, not all ADH1-affected individuals exhibit relative or absolute hypercalciuria (and nephrocalcinosis) (26–28), and this was the case for the proband of the family who was studied here. The proband had basal ganglia calcification and cataracts. Basal ganglia calcification is common in patients with ADH1 (25–30). A mouse strain (designated “Nuf”) harboring an activating CaSR mutation (L723Q) developed ectopic calcification and cataracts (31). The plasma calcium levels in the homozygous Nuf/Nuf mice were more significantly reduced than those in the heterozygous Nuf/+ mice, which were significantly reduced from those in the wild-type mice. Hyperphosphatemia was significantly greater in both the heterozygous and homozygous mice, with a clear trend to higher levels in the homozygous mice than in the heterozygous mice. Also, the heterozygous and homozygous mice were not hypercalciuric (31). In fact, the female Nuf/Nuf mice were hypocalciuric, similar to the proband of the present case. In addition, the ectopic calcification and cataracts were milder in the heterozygous mice than in the homozygous mice (31). Careful evaluation for cataracts should be done in ADH1 cases. With respect to mechanism, the following was suggested (31). The CaSR is expressed in the lens epithelial cells (32), and the elevated intracellular Ca2+ associated with cataract formation (33) triggers the activation of calpain, which modifies cytoskeletal proteins and β-crystallin in lens cataract models (33, 34). Hence, the overactive CaSR mutant may trigger the increases in intracellular Ca2+ that lead to cataract development. CASR R544Q is represented 340 times (as the heterozygote) in public single nucleotide polymorphism databases, with a frequency approaching 0.1%. In the ClinVar database that reports on the relationships among human variations and phenotypes, the eight entries (cases) designated as R544Q cover the spectrum from hypocalcemic to hypercalcemic disorders. In addition, in a single published report, the R544Q variant was found in an individual with familial hypocalciuric hypercalcemia (24). In silico testing assigns the variant as benign (PolyPhen) or tolerated (SIFT). The CaSR couples to intracellular signaling pathways via Gq/11, which leads to increases in intracellular Ca2+, and Gi, which leads to activation of MAPK family members. Our in vitro functional assays examined both of these pathways. In each type of assay, R544Q alone, mimicking the situation in the proband of the present family, was significantly more active than WT in the extracellular Ca2+ dose-response curve. However, R544Q was not more significantly active than WT when coexpressed with WT to mimic the heterozygous state in the parents of the proband. This is fully consistent with the phenotypic expression of R544Q as the homozygote but not as the heterozygote. For comparative purposes, we found that the previously described ADH1 CaSR E604K mutant was significantly more active than the WT alone when evaluated alone and when coexpressed with WT. Moreover, studies with the calcilytic NPS 2143 emphasized the mild overactivity of R544Q relative to the marked overactivity of the E604K mutation. The determination of the crystallographic structure of the CaSR ECD (7) highlighted the important role of the cysteine-rich domain in coupling ligand-induced closure of the VFT domain to the proposed altered conformation of the TMD and interaction with G proteins and signal pathway activation. The cation-π interaction is a stabilizing electrostatic interaction of a cation with the polarizable π electron cloud of an aromatic ring. The cation-π interaction is held to be as important as the hydrophobic effect, the hydrogen bond, or the ion pair in determining macromolecular structure (17, 35). Cation-π interactions have been documented in many proteins in which the arginine or lysine side chains interact with phenylalanine, tyrosine, or tryptophan. The CaSR ECD has five cation-π interactions, and one of these is R544-F539 within the cysteine-rich domain. In the ligand-activated CaSR, the distance between R544 and F539 increases and the electrostatic forces between them weaken. This is consistent with the cation-π interaction contributing to maintenance of the receptor in an inactive conformation when the ligand is absent. Ligand activation overcomes this restraining force, but the cation-π interaction appears to set the potency of the activation, because when it is lost in the R544Q mutant, the potency of activation of the receptor is increased. In our study, we provide further evidence of the nature and importance of the R544-F539 cation-π interaction by the finding that the engineered R544K mutant, which preserves the cationic residue, behaves identically to WT in functional assays, whereas mutant F539A and variant F539L that have lost the aromatic π orbitals are overactive, similar to R544Q. In conclusion, we report on the identification of a rare missense variant in the CaSR cysteine-rich domain that, while not clinically expressed as the heterozygote, came to attention in an individual homozygous for the variant. This has important implications for diagnosis, clinical management, and genetic counseling. Our study also revealed a particular structural feature of the CaSR––a cation-π interaction––that, when lost, makes the receptor more responsive to extracellular calcium. Abbreviations: Abbreviations: ADH1 autosomal dominant hypocalcemia type 1 [Ca2+]o extracellular Ca2+ concentration CASR calcium-sensing receptor gene ECD extracellular domain ExAC Exome Aggregation Consortium GCM2 glial cells missing-2 gene GNA11 G protein α11 gene HEK human embryonic kidney Luc luciferase NFAT nuclear factor of activated T cells NR normal range NS not significant SRE serum response element TMD transmembrane domain VFT Venus flytrap‒like WT wild type Acknowledgments We thank the family members for their participation. We thank NPS Pharmaceuticals, Inc., Salt Lake City, Utah, and Dr. Jenny Yang for HEK293 cells; Drs. Tajima and Fukumoto for promoter-reporter constructs; and Dr. Svetlana Pidasheva and Sarah Elliott for assistance. The authors are also grateful to Nicole Fabien (Service d’Immunologie, UF Autoimunité, Centre Hospitalier, Lyon Sud, France) for the assessment of CaSR antibodies in the proband, to Sara Donato for providing family members’ clinical data, and to Luís Sobrinho for support in the clinical/metabolic interpretation of the case. Financial Support: iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is cofunded by Fundação para a Ciência e Tecnologia/Ministério da Ciência e do Ensino Superior, through national funds, Associação de Endocrinologia Oncológica, Lisboa, Portugal, and FEDER under the PT2020 Partnership Agreement are acknowledged. This study was supported by a Canadian Institutes of Health Research operating grant (to G.N.H.). Disclosure Summary: The authors have nothing to disclose. References 1. Hendy GN , Cole DEC . Familial isolated hypoparathyroidism. In: Brandi ML , Brown EM , eds. Hypoparathyroidism . Milan, Italy: Springer-Verlag Italia ; 2015 : 167 – 175 . 2. 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Published: Aug 1, 2018

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