Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia

Magno, Aaron L.; Leatherbarrow, Kassandra M.; Brown, Suzanne J.; Wilson, Scott G.; Walsh, John P.; Ward, Bryan K.

doi:10.1007/s00223-020-00715-1

Cited by 8 publications

(14 citation statements)

References 36 publications

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“…Patients attending endocrinology clinics at Sir Charles Gairdner Hospital, Perth (patients B1, B2, and C) or the Austin Hospital, Melbourne (patient A) for suspected calcium disorders underwent a full fasting metabolic bone study, which included blood serum examination for ionized calcium; plasma analysis for intact PTH; total calcium, albumin, and creatinine; and either a urine spot (morning, second void) or 24-hour urine test, both of which included calcium and creatinine measurements. Serum, plasma, and urine biochemical analyses were performed using standard methods with calcium to creatinine clearance ratios determined as previously described [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

“…For genetic analysis, genomic DNA extracted from blood samples was subjected to bidirectional Sanger sequencing of segments of the CASR encompassing exons 2 to 7, including splice donor and acceptor sites, to screen the entire coding region. Genetic analysis for patients B1, B2, and C was performed as described previously [ 21 ] while that for patient A was performed by the Molecular Genetics Department, Pathology Queensland, Brisbane, using similar methodologies. Bioinformatics analysis of CASR variants identified by sequence analysis was performed using wANNOVAR software [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

“…Each CASR test variant (T699N, R701G, T808P) was introduced separately by site-directed mutagenesis (Stratagene, La Jolla, CA) into wild-type C-terminally FLAG-tagged human CASR cloned into the mammalian expression vector pcDNA3.1 (CaSR-FLAG/pcDNA3.1) as described previously [ 21 , 23 ]. Transfection-quality plasmid DNA was generated using the Pure Yield miniprep system (Promega Corp, Madison, WI) and quantitated using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA).…”

Section: Methodsmentioning

confidence: 99%

“…All antibodies were from Sigma-Aldrich, St Louis, MO. Bands were visualized using chemiluminescence reagent in a Molecular Imager (Bio-Rad) as described previously [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

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Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia

Mullin

Pavlos

Brown

et al. 2022

Journal of the Endocrine Society

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Background In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcaemia (FHH). Since the conditions may sometimes overlap biochemically, identification of calcium-sensing receptor (CASR) gene variants causative of FHH (but not PHPT) is the most decisive diagnostic aid. When novel variants are identified, bioinformatics and functional assessment are required to establish pathogenicity. Objective We identified three novel CASR transmembrane-domain missense variants, Thr699Asn, Arg701Gly and Thr808Pro, in three probands provisionally diagnosed with FHH and examined the variants using bioinformatics and functional analysis. Methods Bioinformatics assessment utilized wANNOVAR software. For functional characterization, each variant was cloned into a mammalian expression vector and wild type and variant receptors transfected into HEK293 cells and their expression and cellular localization assessed by Western blotting and confocal immunofluorescence, respectively. Receptor activation in HEK293 cells was determined using an IP-One ELISA assay following stimulation with Ca ++ ions. Results Bioinformatics analysis of the variants was unable to definitively assign pathogenicity. Compared with wild type receptor, all variants demonstrated impaired expression of mature receptor reaching the cell surface and diminished activation at physiologically relevant Ca ++ concentrations. Conclusions Three CASR missense variants identified in probands provisionally diagnosed with FHH result in receptor inactivation and are therefore likely causative of FHH. Inactivation may be due to inadequate processing/trafficking of mature receptor and/or conformational changes induced by the variants affecting receptor signalling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcaemic patients.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…All antibodies were from Sigma-Aldrich, St Louis, MO. Bands were visualized using chemiluminescence reagent in a Molecular Imager (Bio-Rad) as described previously [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia

Mullin

Pavlos

Brown

et al. 2022

Journal of the Endocrine Society

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“…Additionally, vitamin D deficiency or impairment of renal function can interfere with Ca 2+ renal excretion [ 52 ]. Therefore, mutational analysis of FHH causative genes and functional studies of derived variants is the gold standard for a proper diagnosis [ 53 , 54 ].…”

Section: Calcium and Its Partnersmentioning

confidence: 99%

The Molecular Basis of Calcium and Phosphorus Inherited Metabolic Disorders

Papadopoulou

Bountouvi

Karachaliou

2021

Genes

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Calcium (Ca) and Phosphorus (P) hold a leading part in many skeletal and extra-skeletal biological processes. Their tight normal range in serum mirrors their critical role in human well-being. The signalling “voyage” starts at Calcium Sensing Receptor (CaSR) localized on the surface of the parathyroid glands, which captures the “oscillations” of extracellular ionized Ca and transfers the signal downstream. Parathyroid hormone (PTH), Vitamin D, Fibroblast Growth Factor (FGF23) and other receptors or ion-transporters, work synergistically and establish a highly regulated signalling circuit between the bone, kidneys, and intestine to ensure the maintenance of Ca and P homeostasis. Any deviation from this well-orchestrated scheme may result in mild or severe pathologies expressed by biochemical and/or clinical features. Inherited disorders of Ca and P metabolism are rare. However, delayed diagnosis or misdiagnosis may cost patient’s quality of life or even life expectancy. Unravelling the thread of the molecular pathways involving Ca and P signaling, we can better understand the link between genetic alterations and biochemical and/or clinical phenotypes and help in diagnosis and early therapeutic intervention.

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Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays

Sörgel

2024

Methods in Molecular Biology

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Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia

Cited by 8 publications

References 36 publications

Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia

Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia

The Molecular Basis of Calcium and Phosphorus Inherited Metabolic Disorders

Analysis of Calcium-Sensing Receptor Signaling Using Dual Luciferase Assays

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