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Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia
CONTEXT: In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (FHH). Since the conditions may sometimes overlap biochemically, identification of calcium-sensing receptor (CASR) gene variants c...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8962451/ https://www.ncbi.nlm.nih.gov/pubmed/35356007 http://dx.doi.org/10.1210/jendso/bvac025 |
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author | Mullin, Benjamin H Pavlos, Nathan J Brown, Suzanne J Walsh, John P McKellar, Ross A Wilson, Scott G Ward, Bryan K |
author_facet | Mullin, Benjamin H Pavlos, Nathan J Brown, Suzanne J Walsh, John P McKellar, Ross A Wilson, Scott G Ward, Bryan K |
author_sort | Mullin, Benjamin H |
collection | PubMed |
description | CONTEXT: In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (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 3 novel CASR transmembrane domain missense variants, Thr699Asn, Arg701Gly, and Thr808Pro, in 3 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; wild-type and variant receptors were transfected into HEK293 cells, and their expression and cellular localization were 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. CONCLUSION: 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 signaling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcemic patients. |
format | Online Article Text |
id | pubmed-8962451 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-89624512022-03-29 Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia Mullin, Benjamin H Pavlos, Nathan J Brown, Suzanne J Walsh, John P McKellar, Ross A Wilson, Scott G Ward, Bryan K J Endocr Soc Clinical Research Article CONTEXT: In the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (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 3 novel CASR transmembrane domain missense variants, Thr699Asn, Arg701Gly, and Thr808Pro, in 3 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; wild-type and variant receptors were transfected into HEK293 cells, and their expression and cellular localization were 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. CONCLUSION: 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 signaling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcemic patients. Oxford University Press 2022-02-18 /pmc/articles/PMC8962451/ /pubmed/35356007 http://dx.doi.org/10.1210/jendso/bvac025 Text en © The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Clinical Research Article Mullin, Benjamin H Pavlos, Nathan J Brown, Suzanne J Walsh, John P McKellar, Ross A Wilson, Scott G Ward, Bryan K Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title | Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title_full | Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title_fullStr | Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title_full_unstemmed | Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title_short | Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia |
title_sort | functional assessment of calcium-sensing receptor variants confirms familial hypocalciuric hypercalcemia |
topic | Clinical Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8962451/ https://www.ncbi.nlm.nih.gov/pubmed/35356007 http://dx.doi.org/10.1210/jendso/bvac025 |
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