Calcium-sensing Receptor Research Articles

Evolutionary history of calcium-sensing receptors unveils hyper/hypocalcemia-causing mutations.

Despite advancements in understanding the structure and functions of the Calcium Sensing Receptor (CaSR), gaps persist in our knowledge of the specific functions of its residues. In this study, we used phylogeny-based techniques to identify functionally equivalent orthologs of CaSR, predict residue significance, and compute specificity-determining position (SDP) scores to understand its evolutionary basis. The analysis revealed exceptional conservation of the CaSR subfamily, emphasizing the critical role of residues with high SDP scores in receptor activation and pathogenicity. To further enhance the findings, gradient-boosting trees were applied to differentiate between gain- and loss-of-function mutations responsible for hypocalcemia and hypercalcemia. Lastly, we investigated the importance of these mutations in the context of receptor activation dynamics. In summary, through comprehensive exploration of the evolutionary history of the CaSR subfamily, coupled with innovative phylogenetic methodologies, we identified activating and inactivating residues, providing valuable insights into the regulation of calcium homeostasis and its connections to associated disorders.

Stone recurrence among Indonesian kidney stone formers: a comprehensive analysis of genetic polymorphism, demographic, and clinical factors

BACKGROUND The recurrence rate of kidney stone disease (KSD) can exceed 50% within 5 years. This study aimed to investigate factors associated with stone recurrence based on single nucleotide polymorphisms (SNPs) of the calcium-sensing receptor (CaSR) rs1801725 and rs1042636, demographic, and clinical profiles. METHODS We collected data on the demographic and clinical characteristics of 80 adult kidney stone formers from April 2021 to September 2022, including peripheral blood samples, morning and 24-hour urine, and 7-day water intake records. Real-time polymerase chain reaction was used to evaluate SNP. Comparisons were made between patients with recurrent and first-time stones. RESULTS 41% of 80 patients have experienced stone recurrence. Recurrent stone formers exhibited significantly higher 24-hour urine calcium excretion (p = 0.03) and lower serum calcium levels (p = 0.019) than first-time stone formers. Hypocitraturia (100%), low urine volume (78%), and hyperoxaluria (55%) were the main abnormalities of all patients. No significant differences were found in CaSR gene polymorphisms and other demographic, biochemical, or clinical parameters. CONCLUSIONS Recurrent stone formers had higher 24-hour urine calcium excretion and lower serum calcium levels. Other risk factors and CaSR polymorphisms may insignificantly affect KSD recurrence.

Detailed functional characterization of four nanobodies as positive allosteric modulators of the human Calcium-Sensing receptor

The calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and small-molecule and peptide positive allosteric modulators (PAMs) of CaSR, so-called calcimimetics, are used in the treatment of hyperparathyroidism and hypocalcemic disorders. In this study, four monovalent nanobodies − representing four distinct nanobody families with CaSR PAM activity − were subjected to elaborate pharmacological profiling at the receptor. While Nb5 displayed negligible PAM activity at CaSR in all assays, Nb4, Nb10 and Nb45 all potently potentiated Ca2+-evoked signalling through a myc epitope-tagged CaSR expressed in HEK293 or HEK293T cells in Gαq and Gαi1 protein activation assays and in a Ca2+/Fluo-4 assay. Nb4 and Nb10 also displayed comparable PAM properties at a stable CaSR-HEK293 cell line in a Ca2+/Fura-2 imaging assay, but surprisingly Nb45 was completely inactive at this cell line in both the Ca2+/Fura-2 and Ca2+/Fluo-4 assays. Investigations into this binary difference in Nb45 activity revealed that the nanobody only possesses modulatory activity at CaSRs tagged N-terminally with various epitopes (myc, HA, Flag-SNAP), whereas it is inactive at the untagged wild-type receptor. In conclusion, overall each of the nanobodies exhibit similar CaSR PAM properties in a range of assays, and thus none of them display pathway bias as modulators. However, of the four nanobodies Nb4 and Nb10 would be applicable as pharmacological tools for the wild-type CaSR, and the complete inactivity of Nb45 at the untagged CaSR serves as an reminder that epitope-tagging of a receptor, even if deemed functionally silent, can have profound implications for ligand discovery efforts.

A Perspective On A Promising Role For Caffeine In Modulating The Casr In Pancreatic Β-Cells

The extracellular calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that plays a crucial role in the parathyroid glands and kidneys in maintaining calcium (Ca2+) homeostasis. The extracellular CaSR is present in various cell types not implicated in controlling plasma Ca2+ levels. There is accumulating evidence that CaSR plays a significant role in regulating the function of β-cells in the pancreas. Divalent cations and insulin are released together during exocytosis, and their concentration in the restricted intercellular compartments of the pancreatic islet increases to activate CaSR in neighboring cells. Subsequently, the activated CaSR stimulates insulin vesicle release in the adjacent cells, and by repeating these steps, the signal is amplified in the whole islet. Recent work developed a photoaffinity-tagged glutathione derivative (DAZ-G) as a chemical tool. The study demonstrated that DAZ-G can identify and analyze CaSR ligands. The use of DAZ-G identified caffeine as a positive allosteric modulator of the CaSR, suggesting that the physiological effects of caffeine are partly attributed to its stimulation of the CaSR. Future work may utilize caffeine to activate the CaSR in pancreatic β-cells to improve their secretory function

CASRdb: A Publicly Accessible Comprehensive Database for Disease-associated Calcium-sensing Receptor Variants.

Genetic testing of the calcium-sensing receptor (CASR) gene is crucial for confirming diagnoses of familial hypocalciuric hypercalcemia type I (FHH1) and autosomal dominant hypocalcemia type I (ADH1). Therefore, we created a publicly accessible comprehensive database of the disease-causing variants of the CASR gene. We used two sources for variant reports: (1) we conducted a systematic review in the Embase and Pubmed databases from inception to March 2023, using search strategies associated with CASR. We identified all articles reporting CASR variants associated with disorders of calcium metabolism. (2) Additionally, data associated with pathogenic (P) or likely pathogenic (LP) variants in the ClinVar and LOVD databases were retrieved. Benign or likely benign variants were excluded. Variants of uncertain significance (VUS) were included only if they were reported in the literature. We generated a library of CASR variants associated with phenotypes, which has been made available on a website. We identified a total of 498 variants, of which 121 (24.3%) were associated with ADH1 and 377 (75.7%) with FHH1. Most included variants were identified from the literature (117 activating and 352 inactivating variants), and the majority of these were not documented in ClinVar/LOVD (73/117, 62.4% activating variants; 207/352, 58.8% inactivating variants). We developed CASRdb, a database that compiles information on all CASR variants associated with disorders of calcium metabolism from existing literature and genomic databases. Our database stands out due to the substantially higher number of disease-associated variants it contains, highlighting its comprehensive nature. The website is available at http://casrdb.mgh.harvard.edu.

Generation of CRISPR/Cas9 modified human iPSC line with correction of heterozygous mutation in exon 6 of the CaSR gene.

The calcium-sensing receptor (CaSR) gene encodes a cell membrane G protein-coupled receptor (GPCR) which has a key role in maintaining the extracellular Ca2+ homeostasis. We aimed at correcting the compound heterozygous mutation in the 6th [c.1656delA, p.I554SfsX73] and 7th [c.2217T > A, p.C739X] exons of the CASR gene which the original patient-derived iPSC line had. The mutation is associated with neonatal severe primary hyperparathyroidism of the patient. We generated and characterized a CRISP/Cas9-edited hiPSC line with the restored sequence in the sixth exon of the CASR gene, bearing only heterozygous mutation in the 7th exon. The results showed that the new genetically modified cell line has karyotype without abnormalities, typical hiPSCs morphology, characteristic expression of pluripotency markers, and ability to develop into three germ layers, and differentiates in chondrogenic, adipogenic, osteogenic directions. This new cell line will complement the existing pool of CaSR-mutated cell lines, a valuable resource for in-depth understanding of neonatal severe primary hyperparathyroidism. This will allow further exploration of the application of pharmacological drugs in the context of personalized medicine to correct Ca-homeostasis disorders.

Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels. CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca2+ using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing. RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca2+ elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca2+ responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids. The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.

Amelioration of Type 2 Diabetes Mellitus Using Rapeseed (Brassica napus)-Derived Peptides through Stimulating Calcium-Sensing Receptor: Effects on Glucagon-Like Peptide-1 Secretion and Hepatic Lipid Metabolism.

The potential of rapeseed-derived peptides (RDPs) in the amelioration of type 2 diabetes mellitus (T2DM) has been hypothesized. However, the mechanisms of the intestinal endocrine hormones activated by RDPs have not been fully understood. This study aimed to explore the amelioration of T2DM and associated hepatic lipid metabolism disorders using RDPs by affecting glucagon-like peptide-1 (GLP-1) secretion. Eight RDPs were prepared by different stepwise enzymatic hydrolysis, wherein RCPP-3 (sequential using alcalase/flavourzyme) and RNPP-1 (sequential using alcalase/trypsin) maintained the normal blood glucose level, significantly increased the body weight (27.17 ± 0.19%) in T2DM mice compared to the positive group (p < 0.05). Western blotting and immunofluorescence experiments indicated that RCPP-3 and RNPP-1 regulated the intestinal endocrine hormones GLP-1 secretion through the calcium-sensing receptor (CaSR). Additionally, the PI3K-Akt pathway was significantly activated by GLP-1, leading to marked improvements in hepatic lipid parameters (TC, TG, LDL-c, and HDL-c) and mitigated fat accumulation (p < 0.05). Notably, the stimulating effect of RCPP-3 on GLP-1 was 10.05% ± 0.71% higher than RNPP-1. G2-R3, a fraction separated from RCPP-3, which contained 14 peptides with the best capacity to stimulate GLP-1 secretion, was identified using HPLC-QTOF-MS/MS. This study suggests the potential of RDPs as novel functional food supplements for ameliorating T2DM.

Early divergent modulation of NLRP2′s and NLRP3′s inflammasome sensors vs. AIM2′s one by signals from Aβ·Calcium-sensing receptor complexes in human astrocytes

Alzheimer’s disease (AD), the most prevalent human dementia, is driven by accruals of extracellular Aβ42 senile patches and intracellular neurofibrillary tangles of hyperphosphorylated Tau (p-Tau) proteins. AD’s concurrent neuroinflammation is prompted by innate immunity-related cytosolic protein oligomers named inflammasomes. Upon proper “first” (priming) and “second” (activating) signals, inflammasomes overproduce proinflammatory Interleukin (IL)-1β, and IL-18 while cleaving pyroptosis-promoting Gasdermin D’s N-terminal fragments. Our earlier studies highlighted that in pure monocultures, exogenous Aβ25-35-treated nonproliferating human cortical astrocytes (HCAs) made and released surpluses of endogenous Aβ42-oligomers (−os) and p-Tau-os, just as alike-treated human cortical neurons did. Aβ25-35-exposed HCAs also over-released NO, VEGFA, and IL-6. Aβ•CaSR (Aβ·Calcium-Sensing Receptor) complexes generated intracellular signals mediating all such neurotoxic effects since CaSR’s negative allosteric modulators (aka NAMs or calcilytics, e.g., NPS2143) fully suppressed them. However, it had hitherto remained unexplored whether signals from Aβ·CaSR complexes also induced the early expression and/or activation of NOD-like 2 (NLRP2) and 3 (NLRP3) and of PYHIN absent in melanoma 2 (AIM2) inflammasomes in monocultured HCAs. To clarify this topic, we used in-situ-Proximity Ligation, qRT-PCR, double antibody arrays, immunoblots, and Caspase 1/4 enzymatic assays. Aβ·CaSR complexes quickly assembled on HCAs surface and issued intracellular signals activating Akt and JAK/STAT axes. In turn, the latter upregulated NLRP2 and NLRP3 PRRs (pattern recognition receptors) yet downregulated AIM2. These effects were specific, being significantly hindered by NPS2143 and inhibitors of PI3K (LY294002), AMPKα (Dorsomorphin), mTOR (Torin1), and JAK/TYK (Brepoticinib). A wide-spectrum inhibitor, Bay11-7082, intensified the Aβ·CaSR/Akt/JAK/STAT axis-driven opposite control of NLRP3’s and AIM2’s PRR proteins without affecting NLRP2 PRR upregulation. However, the said effects on the PRRs proteins vanished within 24-h. Moreover, Aβ·CaSR signals neither concurrently changed ASC, pro-IL-1β, and Gasdermin-D (holo- and fragments) protein levels and Caspases 1 and 4 enzymatic activities nor induced pyroptosis. Therefore, Aβ·CaSR cues acted as “first (priming) signals” temporarily increasing NLRP2 and NLRP3 PRRs expression without activating the corresponding inflammasomes. The neatly divergent modulation of NLRP3’s vs. AIM2’s PRR proteins by Aβ·CaSR cues and by Bay11-7082 suggests that, when bacterial or viral DNA fragments are absent, AIM2 might play “anti-inflammasomal” or other roles in HCAs. However, Bay11-7082’s no effect on NLRP2 PRR overexpression also reveals that CaSR’s downstream mechanisms controlling inflammasomes’ sensors are quite complex in HCAs, and hence, given AD’s impact on human health, well worth further studies.

Heterogenous Origins of Calcium Homeostasis Disorders Arising from Five Heterozygous Calcium-Sensing Receptor Variants.

The human calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and most identified CASR variants are associated with hypercalcemic and hypocalcemic disorders. Here we characterized the pharmacological implications of five heterozygous CASR variants from individuals with familial hypocalciuric hypercalcemia 1 [FHH1: Y63C, I81T, Q459R, W818stop] or autosomal dominant hypocalcemia 1 [ADH1: R955stop]. Total and cell surface expression levels of wild-type (WT) and variant CaSRs expressed in human embryonic kidney 293T (HEK293T) cells were determined using ELISA, and the pharmacological properties of the receptors were delineated in two functional assays. The Y63C and I81T variations in the extracellular domain (ECD) of CaSR yielded markedly reduced cell surface expression and Ca2+ responsiveness, while Q459R displayed WT-like expression and functional properties. Truncation of the 7-transmembrane domain (7TMD) in W818stop eliminated cell surface expression, whereas R955stop in the intracellular carboxy-terminal yielded modestly increased surface expression and Ca2+ potency compared with WT CaSR. Interestingly, the effectiveness of positive allosteric modulators (PAMs) at the variants varied. Ca2+-mediated signaling through Y63C and I81T was significantly augmented by 7TMD-binding PAMs (NPS R-568 and Evocalcet) but not by ECD-binding PAMs (Etelcalcetide and Nb4), whereas signaling through Q459R and R955stop were robustly potentiated by all four PAMs. While the molecular phenotypes exhibited by the five CaSR variants concord with the clinical phenotypes in individuals harboring them, CASR variant-induced calcium homeostasis disorders clearly arise from diverse molecular origins, and the effectiveness of calcimimetics in these disorders could differ depending on the specific variants.

In silico analysis of the melamine structural analogues interaction with calcium-sensing receptor: A potential for nephrotoxicity

In recent years, melamine, and its structural analogues, as adulterants in various food products including protein supplements,have been widely studied for their nephrotoxic effects. Previous research has presented evidence that certain small molecules can alter the calcium-sensing receptor (CaSR) function, contributing to nephrotoxicity. Melamine, for example, has been observed in in vitro settings to interact with the allosteric binding site of CaSR, resulting in uncontrolled CaSR activation. This activation results in the production of reactive oxygen species, which eventually causes kidney cell apoptosis and/or necrosis. The present research used the in silico molecular modelling to evaluate the CaSR binding profilesof four common adulterants in protein supplements: melamine, cyanuric acid, uric acid, and melamine cyanurate. Using Schrödinger’s Maestro docking software (version 13.2.128), the docking studies coupled a noncovalent extra precision mode with the molecular mechanics-generalized born surface area (MM-GBSA) simulation for enhanced binding affinity prediction accuracy. This study identified that cyanuric acid, uric acid, and melamine cyanurate have greater CaSR binding affinities than melamine. Interestingly, melamine cyanurate had the highest binding potential to CaSR. Previous animal studies have reported high concentrations of melamine cyanurate complex in rat kidneys following melamine administration. These findings demonstrate a molecular explanation melamine cyanurate complex-induced nephrotoxicity. This research offers new insight regarding the probable mechanism through which melamine, its analogues, and complexes may cause nephrotoxicity.

New calcimimetics for secondary hyperparathyroidism in CKD G5D: do they offer advantages?

Secondary hyperparathyroidism is one of the most frequent metabolic abnormalities found in patients with chronic kidney disease. The calcium-sensing receptor senses extracellular calcium and is the principal regulator of parathyroid hormone secretion. Cloning of the calcium-sensing receptor led to the development of calcimimetics, drugs that decrease parathyroid hormone secretion through the positive allosteric modulation of this receptor. Cinacalcet was the first oral calcimimetic approved by the US Food and Drug Administration (FDA) in 2004 for the treatment of secondary hyperparathyroidism in adult patients on dialysis. Although cinacalcet has demonstrated safety and effectiveness, it has two main problems: gastrointestinal side effects that result in poor adherence, and the inhibitory action on CYP2D6 with the possibility of interactions with commonly used medications. To address the problem of oral compliance, Etelcalcetide, a small synthetic polycationic peptide IV calcimimetic was introduced in 2017. This drug showed a 10% greater decrease in serum parathyroid hormone values compared to cinacalcet but no better gastrointestinal tolerance, with greater risk of hypocalcemia. Several structural modifications were introduced in cinacalcet to produce a new compound called evocalcet. This drug, which was introduced in Japan in 2018, has considerably enhanced bioavailability and decreased both the inhibitory effect on CYP2D6 and half of the gastrointestinal side effects of cinacalcet. Finally, a novel non-peptidic injectable calcimimetic agent, upacicalcet, became available in Japan in 2021. This agent has greater clearance by hemodialysis and shows no effect on gastric emptying. More studies are needed comparing the old calcimimetics to the new ones to establish their future role in the treatment of secondary hyperparathyroidism in chronic kidney disease (CKD) G5D.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays.

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene.

Measuring IP3 Generation in Real-Time Using a NanoBiT Luminescence Biosensor.

G protein-coupled receptors that activate Gq/11 regulate a range of physiological processes including neurotransmission, energy homeostasis, blood pressure regulation, and calcium homeostasis. Activation of Gq/11-coupled receptors stimulates the generation of inositol 1,4,5-trisphosphate (IP3), which mobilizes intracellular calcium release from the endoplasmic reticulum. This chapter describes an assay that uses a NanoBiT-IP3 luminescent biosensor to detect increases in IP3 in live cells. It describes how to perform these assays to assess signaling by the ghrelin receptor and the calcium-sensing receptor in HEK293 cells.

7331 A Rare Case Of Familial Hypoparathyroidism

Abstract Disclosure: N. Sekhon: None. K.Z. Win: None. Background: Autosomal dominant hypoparathyroidism disorders are very rare and mostly asymptomatic. We present an interesting case of autosomal hypoparathyroidism with a new variant of gene defect on genetic analysis. Case: A 66-year-old female was referred to the endocrinology clinic for familial hypoparathyroidism and calcium oxalate nephrolithiasis. She had onset of seizures at age 16. She was diagnosed with hypocalcemia and hypoparathyroidism at age 30 when she had her first episode of nephrolithiasis. She had recurrent nephrolithiasis and lithotripsies, ultimately developing chronic kidney impairment that was at stage 3b at the time of evaluation. Renal Ultrasound revealed bilateral nephrolithiasis, and nephrocalcinosis. There was a history of hypoparathyroidism in her father, paternal uncle, and son. Her medications included calcium carbonate-vitamin D3 600-400 mg-unit oral 2 tablets daily and calcitriol 0.5 mcg daily. The pertinent physical examination finding was a short stature with height 4’11” and calluses in fingers. Her recent serum chemistry showed calcium 8.8 mg/dL (reference range 8.3 - 10.6 mg/dL), PTH &amp;lt; 6.3 pg/mL (reference range 18.4 - 80.1 pg/ mL), and phosphorus 3.3 mg/dL (reference range 2.4 - 5.5 mg/dL). 24 hr urine calcium was 96 mg (reference range 50-300 mg/24h). She was treated with hydrochlorothiazide for nephrolithiasis but was discontinued due to hypokalemia. We performed genetic testing that resulted positive for CASR NM_001178065.1 with heterozygosity and damaging missense predictions inherited as autosomal dominant or recessive. Discussion: The calcium-sensing receptor (CaSR) has an important role in calcium homeostasis by regulating parathyroid hormone (PTH) secretion and urinary calcium excretion. Familial hypoparathyroidism disorders are divided into Autosomal dominant hypocalcemia (ADH) type 1 and Autosomal dominant hypocalcemia (ADH) type 2. ADH1 is caused by an activating mutation of calcium-sensing receptor (CASR). The estimated prevalence of ADH1 ranges from 1 per 70,000 to 3.9 per 100,000. It is characterized by mild to moderate hypocalcemia, hyperphosphatemia, hypercalciuria, and inappropriately low but detectable PTH levels. It can be associated with Bartter syndrome Type 5. ADH2 is due to mutations in the alpha subunit of the G protein G11 (Gα11). Our patient had classic symptoms of hypocalcemia including short stature, seizures, kidney stones and its sequelae. Her 24-hour urine analysis showed inappropriately normal urine calcium with low normal serum calcium, undetectable serum PTH, and normal phosphorus. Our patient’s genetic analysis revealed a heterozygous defect in gene CASR with DNA variants c.2564G&amp;gt;A with damaging missense predictions. There is no such variant reported or listed in the ClinVar database. To our knowledge, this is the first such case of an autosomal dominant hypoparathyroidism with this gene defect. Presentation: 6/1/2024

12520 Preliminary Findings From An Ongoing Open-label Phase 2 Study Demonstrate That The Calcilytic Encaleret Can Improve The Relationship Between Blood And Urinary Calcium In Individuals With Post-surgical Hypoparathyroidism

Abstract Disclosure: I.R. Hartley: Research Investigator; Self; Calcilytix Therapeutics, Inc. R.I. Gafni: Research Investigator; Self; Calcilytix Therapeutics, Inc. K.L. Roszko: Research Investigator; Self; Calcilytix Therapeutics, Inc. X. Li: None. E.A. Ferguson: Research Investigator; Self; Calcilytix Therapeutics, Inc. C.A. Moore: Research Investigator; Self; Calcilytix Therapeutics, Inc. K.A. Pozo: Research Investigator; Self; Calcilytix Therapeutics, Inc. K.T. Ampuero: Research Investigator; Self; Calcilytix Therapeutics, Inc. A.V. Sridhar: Employee; Self; Calcilytix Therapeutics, Inc. A.S. Mathew: Employee; Self; Calcilytix Therapeutics, Inc. M. Roberts: Employee; Self; Calcilytix Therapeutics, Inc. S.H. Adler: Employee; Self; Calcilytix Therapeutics, Inc. E.F. Nemeth: Consulting Fee; Self; Calcilytix Therapeutics, Inc. M.T. Collins: Research Investigator; Self; Calcilytix Therapeutics, Inc. Parathyroid hormone (PTH) and the calcium-sensing receptor (CaSR) are the primary regulators of blood and urinary calcium. The independent contributions of PTH and the CaSR on renal calcium handling are not well understood, largely due to their intertwined physiology. In individuals with functioning parathyroid tissue, calcilytics (negative modulators of the CaSR) increase PTH secretion and decrease urinary calcium excretion, leading to increased blood calcium levels. We hypothesized that calcilytic administration to individuals with post-surgical hypoparathyroidism (PSH) may reveal the PTH-independent effects of CaSR modulation on renal calcium handling and clarify the potential therapeutic role of calcilytics in PSH. Four women (26-69y) with PSH enrolled in an ongoing, open-label, phase 2, proof-of-principle study of encaleret, an oral investigational calcilytic (NCT05735015). Encaleret 162 mg was administered every 12 hours for up to 10 doses. Calcitriol was discontinued one day prior to the first dose of encaleret. All participants received calcium supplementation initially. To maintain eucalcemia, 1 participant discontinued calcium supplementation and 3 required re-initiation of low-dose calcitriol (0.25 mcg daily). Results are presented as mean (range) of fasting baseline pre-dose levels compared to fasting levels 12 hours after the last dose of encaleret. Fractional excretion of calcium (FECa), a measure of renal calcium handling that quantifies the relationship between blood and urinary calcium, decreased by 52% (34-78). Albumin-corrected blood calcium increased from 8.4 mg/dL (range 8.1-8.6; nl 8.4-10.2) to 9.3 (8.6-10.7). Urinary calcium excretion decreased from 348 mg/day (range 204-501; nl &amp;lt;250) to 169 (76-350), with normalization in 3 participants and a 30% decrease in the fourth. Baseline PTH of 7.4 pg/mL (range 4.6-10.1; nl 15-65) transiently increased 30 minutes after the first dose to 13.0 (4.5-18.1), then returned to near baseline levels. Bone turnover markers were normal and did not change on encaleret. There were no serious adverse events reported. The only treatment-related adverse events (AEs) were mild hypercalcemia causing headache in 1 participant. Her blood calcium levels continued to be mildly elevated for 60 hours after the last dose of encaleret even after calcium and calcitriol supplementation was discontinued. Despite elevated blood calcium, her 24-hour urine calcium remained &amp;lt;200 mg/day. Encaleret reduced FECa in the first four participants with PSH, improving the relationship between blood and urinary calcium. These preliminary results from this Phase 2 study support continued evaluation of encaleret as an orally administered therapy for the treatment of patients with PSH. Presentation: 6/1/2024

7633 Novel CASR Gene Mutation in a patient with Familial Hypocalciuric Hypercalcemia and Papillary Thyroid Cancer

Abstract Disclosure: A. Parihar: None. E.S. Nylen: None. S.M. Gandhi: None. Familial Hypocalciuric Hypercalcemia (FHH) is predominantly caused by loss-of-function mutations in the calcium-sensing receptor (CaSR) characterized by a benign elevation in calcium and PTH which can be mistaken for primary hyperparathyroidism (PHPT). Missense mutations in the CaSR gene causing FHH have been documented but not much is known regarding their clinical significance. We describe a patient with FHH found to have a novel variant mutation. A 37-year-old male with hypogonadism and fatty liver was incidentally found to have hypercalcemia suggestive of PHPT with elevated PTH (102-114 pg/ml), normal vitamin D, and Fractional Excretion of Calcium (FECa) of 0.014. His father also had hypercalcemia and had undergone parathyroid surgery. The patient denied kidney stones and had a normal bone density. Subsequent Sestamibi and 4D CT scan suggested parathyroid adenoma on the left mid-lower pole of the thyroid. Neck ultrasound also showed a left 1.2 x 1 x 1 cm thyroid nodule as well as a left neck mass with calcifications. Fine needle aspiration was positive for metastatic Papillary thyroid cancer(PTC) in the left neck mass and patient underwent total thyroidectomy along with subtotal parathyroidectomy and lateral neck dissection. Thyroid pathology confirmed multifocal PTC with lymph node involvement that were BRAF V600 positive. Parathyroid pathology was benign hyperplasia. The PTH level decreased appropriately intraoperatively (69%), but postoperatively a rise was noted with levels remaining 70-90 pg/ml with normal calcium levels. Repeat FECa was 0.007, supporting the diagnosis of FHH. Given patient’s father also had hypercalcemia, genetic testing was pursued. This revealed CaSR gene missense mutation (c.1382T&amp;gt;C p.Leu461Pro, heterozygous). This variation is not present in population databases and has been classified as a variant of uncertain significance. It can be difficult to distinguish PHPT from FHH based on FECa as in this case. The patient also had PTC which is not known to be associated with FHH. Due to uncertain significance of this novel CaSR missense mutation, long term follow-up and further familial genetic testing will help better understand clinical implications. Presentation: 6/2/2024

8082 Primary Hyperparathyroidism or Familial Hypocalciuric Hypercalcemia? Why Not Both?

Abstract Disclosure: C.A. Zmijewski: None. P.D. Greenberg: None. S.R. Saul: None. Background: Although considered two separate entities, primary hyperparathyroidism (PHPT) and familial hypocalciuric hypercalcemia (FHH) can present concurrently. Case: A 68-year-old male with hypertension was referred to Endocrinology for hypercalcemia and osteoporosis. The patient reported a long-standing history of intermittent hypercalcemia, first diagnosed at age 30 with a strong family history, both his father and son. He denied symptoms of hypercalcemia, history of nephrolithiasis and fractures, and was not taking calcium supplementations. Labs were notable for intermittent elevations in calcium, the highest being 10.9 mg/dL (8.5-10.5 mg/dL), in addition to phosphorus levels as low as 1.7 mg/dL (2.5-4.5 mg/dL). Vitamin D was replete at 50 ng/mL (32-100 ng/mL) and the highest parathyroid hormone (PTH) was 124 pg/mL (10-66 pg/mL). The 24-hour urine for calcium was 117 mg (100-300 mg) with a Ca/Cr 0.01. DEXA scan showed T scores -2.2 at the right femoral neck and -3.4 at the distal radius (Z score -2.3). Due to the significant osteoporosis, a work-up for secondary causes was completed and was unremarkable. The differential diagnosis for the osteoporosis, elevated parathyroid hormone, and intermittent hypercalcemia included PHPT vs FHH or possibly, a combination. Additional imaging included a renal ultrasound which was negative for nephrolithiasis, Sestamibi and 4D-CT which were both negative for a parathyroid adenoma. Genetic testing confirmed a mutation in the CASR gene. Due to the inability to localize a possible lesion, the decision was made to treat the osteoporosis with zoledronic acid. Conclusion: PHPT is a common endocrine disorder; approximately 100,000 people in the United States develop PHPT annually. Conversely, FHH is a rare endocrine disorder with an incidence rate of 1 in 78,000. PHPT occurs due to the over-secretion of PTH by one or more parathyroid glands. FHH is an inherited autosomal dominant disorder, most commonly due to an inactivating mutation in the calcium-sensing receptor (CASR) gene. Distinguishing PHPT from FHH is important because PHPT is cured with surgery while FHH does not require treatment. Hypercalcemia from FHH, as opposed to PHPT, is suggested by the absence of symptoms, a positive family history, normal or high PTH, and hypocalciuria. However, it can be challenging to separate the two especially in atypical presentations (PTH can be normal in PHPT and elevated in FHH, and Ca/Cr can be &amp;lt; 0.01 in PHPT). But what if these two conditions were to co-exist? Case reports have described patients with concurrent PHPT and FHH (parathyroid adenoma confirmed with pathology and CASR gene mutation confirmed with genetic testing). In these cases, parathyroidectomy can still be considered in co-existing PHPT and FHH to alleviate symptoms and prevent complications of hypercalcemia. Though a rare occurrence, PHPT and FHH have been shown to co-exist in prior case reports. Presentation: 6/1/2024