Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

Identification of variants in the calcium-sensing receptor (CASR) gene is an important means of distinguishing between familial hypocalciuric hypercalcaemia (FHH) and primary hyperparathyroidism. However, identification and bioinformatics analysis of genetic variants alone is now considered insufficient as definitive proof; additional functional assessment is required to diagnose FHH with certainty. We identified two novel variants, D433Y and C739Y, and one previously reported variant G509R in the CASR of four kindreds provisionally diagnosed with FHH and aimed to functionally characterise these variants to confirm the diagnosis. Variant receptors were cloned as FLAG-tagged constructs into the mammalian expression vector, pcDNA3.1. Wild type and variant receptor constructs were expressed in HEK293 cells and their expression assessed by Western blot analysis and their functionality analysed using an IP-One assay which measures myo-inositol 1-phosphate accumulation following CaSR activation. Western blot analysis showed that the D433Y receptor had diminished mature glycosylated receptor compared with wild type CaSR whereas the G509R receptor had a complete lack of mature receptor. The C739Y receptor was consistently overexpressed. Functional assessment showed the D433Y receptor to be mildly inactivating at physiological calcium concentrations whereas the G509R receptor was inactive at all calcium concentrations. By contrast, the C739Y variant was activating compared to wild type receptor which is inconsistent with it causing FHH. We conclude that functional assessment of CaSR variants using the IP-One assay was useful in the investigation of suspected FHH probands, confirming the D433Y and G509R variants as likely pathogenic/pathogenic, but dismissing the C739Y variant as causing FHH.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Lee JY, Shoback DM (2018) Familial hypocalciuric hypercalcemia and related disorders. Best Pract Res Clin Endocrinol Metab 32:609–619

    Article  CAS  Google Scholar 

  2. Ward BK, Magno AL, Walsh JP, Ratajczak T (2012) The role of the calcium-sensing receptor in human disease. Clin Biochem 45:943–953

    Article  CAS  Google Scholar 

  3. Jakobsen NFB, Rolighed L, Moser E, Nissen PH, Mosekilde L, Rejnmark L (2014) Increased trabecular volumetric bone mass density in familial hypocalciuric hypercalcaemia (FHH) type 1: a cross sectional study. Calcif Tissue Int 95:141–152

    Article  CAS  Google Scholar 

  4. Isaksen T, Nielsen CS, Christensen SE, Nissen PH, Heickendorff L, Mosekilde L (2011) Forearm bone mineral density in familial hypocalciuric hypercalcaemia and primary hyperparathyroidism: a comparative study. Calcif Tissue Int 89:285–294

    Article  CAS  Google Scholar 

  5. Magno AL, Ward BK, Ratajczak T (2011) The calcium-sensing receptor: a molecular perspective. Endocr Rev 32:3–30

    Article  CAS  Google Scholar 

  6. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC (1993) Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366:575–580

    Article  CAS  Google Scholar 

  7. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, Seidman JG (1993) Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 31:1297–1303

    Article  Google Scholar 

  8. Hendy GN, Guarnieri V, Canaff L (2009) Calcium-sensing receptor and associated diseases. Prog Mol Biol Transl Sci 89:31–95

    Article  CAS  Google Scholar 

  9. Gorvin CM (2019) Molecular and clinical insights from studies of calcium-sensing receptor mutations. J Mol Endocrinol 63:R1–R16. https://doi.org/10.1530/JME-19-0104

    Article  CAS  PubMed  Google Scholar 

  10. Nesbit MA, Hannan FM, Howles SA, Babinsky VN, Head RA, Cranston T, Rust N, Hobbs MR, Heath H III, Thakker RV (2013) Mutations affecting G-protein subunit α11 in hypercalcemia and hypocalcemia. N Engl J Med 368:2476–2486. https://doi.org/10.1056/NEJMoa1300253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nesbit MA, Hannan FM, Howles SA, Reed AAC, Cranston T, Thakker CE, Gregory L, Rimmer AJ, Rust N, Graham U, Morrison PJ, Hunter SJ, Whyte MP, McVean G, Buck D, Thakker RV (2013) Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3. Nat Genet 45:93–97. https://doi.org/10.1038/ng.2492

    Article  CAS  PubMed  Google Scholar 

  12. Stuckey BGA, Kent GN, Gutteridge DH, Pullan PT, Price RI, Bhagat C (1987) Fasting calcium excretion and parathyroid hormone together distinguish familial hypocalciuric hypercalcaemia from primary hyperparathyroidism. Clin Endocrinol 27:525–533

    Article  CAS  Google Scholar 

  13. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG (1994) Autosomal dominant hypocalcaemia caused by a Ca2+-sensing receptor gene mutation. Nat Genet 8:303–307

    Article  CAS  Google Scholar 

  14. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17:405–424

    Article  Google Scholar 

  15. Ward BK, Magno AL, Blitvich BJ, Rea AJ, Stuckey BGA, Walsh JP, Ratajczak T (2006) Novel mutations in the calcium-sensing receptor gene associated with biochemical and functional differences in familial hypocalciuric hypercalcaemia. Clin Endocrinol 64:580–587

    Article  CAS  Google Scholar 

  16. Nordin BEC (1978) Diagnostic procedures in disorders of calcium metabolism. Clin Endocrinol 8:55–67

    Article  CAS  Google Scholar 

  17. Ward BK, Magno AL, Davis EA, Hanyaloglu AC, Stuckey BGA, Burrows M, Eidne KA, Charles AK, Ratajczak T (2004) Functional deletion of the calcium-sensing receptor in a case of neonatal severe hyperparathyroidism. J Clin Endocrinol Metab 89:3721–3730

    Article  CAS  Google Scholar 

  18. Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38:e164. https://doi.org/10.1093/nar/gkq603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cunningham F, Achuthan P, Akanni W, Allen J, Amode MR, Armean IM, Bennett R, Bhai J, Billis K, Boddu S, Cummins C, Davidson C, Dodiya KJ, Gall A, Giron CG, Gil L, Grego T, Haggerty L, Haskell E, Hourlier T et al (2019) Ensembl 2019. Nucleic Acids Res 47(Database issue):D745–D751. https://doi.org/10.1093/nar/gky1113

    Article  CAS  PubMed  Google Scholar 

  20. Yang H, Robinson PN, Wang K (2015) Phenolyzer: phenotype-based prioritization of candidate genes for human diseases. Nat Methods 12:841–843. https://doi.org/10.1038/nmeth.3484

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Arulpragasam A, Magno AL, Ingley E, Brown SJ, Conigrave AD, Ratajczak T, Ward BK (2012) The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling. Biochem J 441:995–1006

    Article  CAS  Google Scholar 

  22. Ward BK, Stuckey BG, Gutteridge DH, Laing NG, Pullan PT, Ratajczak T (1997) A novel mutation (L174R) in the Ca2+-sensing receptor gene associated with familial hypocalciuric hypercalcemia. Hum Mutat 10:233–235

    Article  CAS  Google Scholar 

  23. Nissen PH, Christensen SE, Heickendorff L, Brixen K, Mosekilde L (2007) Molecular genetic analysis of the calcium sensing receptor gene in patients clinically suspected to have familial hypocalciuric hypercalcemia: phenotypic variation and mutation spectrum in a Danish population. J Clin Endocrinol Metab 92:4373–4379

    Article  CAS  Google Scholar 

  24. Geng Y, Mosyak L, Kurinov I, Zuo H, Sturchler E, Cheng TC, Subramanyam P, Brown AP, Brennan SC, Mun H, Bush M, Chen Y, Nguyen TX, Cao B, Chang DD, Quick M, Conigrave AD, Colecraft HM, McDonald P, Fan QR (2016) Structural mechanism of ligand activation in human calcium-sensing receptor. eLife 5:e13662. https://doi.org/10.7554/eLife.13662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Vargas-Poussou R, Mansour-Hendili L, Baron S, Bertocchio J, Travers C, Simian C, Treard C, Baudouin V, Beltran S, Broux F, Camard O, Cloarec S, Cormier C, Debussche X, Dubosclard E, Eid C, Haymann J, Kiando SR, Kuhn J, Lefort G et al (2016) Familial hypocalciuric hypercalcemia types 1 and 3 and primary hyperparathyroidism: similarities and differences. J Clin Endocrinol Metab 101:2185–2195

    Article  CAS  Google Scholar 

  26. Murugaian EE, Premkumar RMR, Radhakrishnan L, Vallath B (2008) Novel mutations in the calcium sensing receptor gene in tropical chronic pancreatitis in India. Scand J Gastroenterol 43:117–121

    Article  CAS  Google Scholar 

  27. Ray K, Clapp P, Goldsmith PK, Spiegel AM (1998) Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. J Biol Chem 273:34558–34567

    Article  CAS  Google Scholar 

  28. Kulkarni A, Mohite M, Vijaykumar R, Bansode P, Murade S, Tamhankar PM (2014) Neonatal severe hyperparathyroidism due to compound heterozygous mutation of calcium sensing receptor (CaSR) gene presenting as encephalopathy. Indian J Pediatr 81:1228–1229

    Article  Google Scholar 

  29. Ho C, Conner DA, Pollak MR, Ladd DJ, Kifor O, Warren HB, Brown EM, Seidman JG, Seidman CE (1995) A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nat Genet 11:389–394

    Article  CAS  Google Scholar 

  30. Lienhardt A, Garabedian M, Bai M, Sinding C, Zhang Z, Lagarde J, Boulesteix J, Rigaud M, Brown EM, Kottler M (2000) A large homozygous or heterozygous in-frame deletion within the calcium-sensing receptor's carboxylterminal cytoplasmic tail that causes autosomal dominant hypocalcemia. J Clin Endocrinol Metab 85:1695–1702

    CAS  PubMed  Google Scholar 

  31. Ikegaki N, Gotoh T, Kung B, Riceberg JS, Kim DY, Zhao H, Rappaport EF, Hicks SL, Seeger RC, Tang XX (2007) De novo identification of MIZ-1 (ZBTB17) encoding a MYC-interacting zinc-finger protein as a new favorable neuroblastoma gene. Clin Cancer Res 13:6001–6009

    Article  CAS  Google Scholar 

  32. Birnbaum RY, Clowney EJ, Agamy O, Kim MJ, Zhao J, Yamanaka T, Pappalardo Z, Clarke SL, Wenger AM, Nguyen L, Gurrieri F, Everman DB, Schwartz CE, Birk OS, Bejerano G, Lomvardas S, Ahituv N (2012) Coding exons function as tissue-specific enhancers of nearby genes. Genome Res 22:1059–1068

    Article  CAS  Google Scholar 

  33. Ray K, Hauschild BC, Steinbach PJ, Goldsmith PK, Hauache O, Spiegel AM (1999) Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca2+ receptor critical for dimerization. J Biol Chem 274:27642–27650

    Article  CAS  Google Scholar 

  34. Ray K, Ghosh SP, Northup JK (2004) The role of cysteines and charged amino acids in the extracellular loops of the human Ca2+ receptor in cell surface expression and receptor activation processes. Endocrinology 145:3892–3903

    Article  CAS  Google Scholar 

  35. Guarnieri V, Canaff L, Yun FHJ, Scillitani A, Battista C, Muscarella LA, Wong BYL, Notarangelo A, D'Agruma L, Sacco M, Cole DEC, Hendy GN (2010) Calcium-sensing receptor (CASR) mutations in hypercalcemic states: studies from a single endocrine clinic over three years. J Clin Endocrinol Metab 95:1819–1829

    Article  CAS  Google Scholar 

  36. Marx SJ (2019) New concepts about familial isolated hyperparathyroidism. J Clin Endocrinol Metab 104:4058–4066

    Article  Google Scholar 

  37. Ong GSY, Walsh JP, Stuckey BGA, Brown SJ, Rossi E, Ng JL, Nguyen HH, Kent GN, Lim EM (2012) The importance of measuring ionized calcium in characterizing calcium status and diagnosing primary hyperparathyroidism. J Clin Endocrinol Metab 97:3138–3145

    Article  CAS  Google Scholar 

Download references

Funding

This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author information

Author notes

  1. Aaron L. Magno and Kassandra M. Leatherbarrow should be considered joint first author.

Authors and Affiliations

  1. Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Block C, Level 1, Hospital Avenue, Nedlands, WA, Australia

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

  2. School of Biomedical Sciences, University of Western Australia, Nedlands, WA, Australia

    Scott G. Wilson

  3. Department of Twin Research and Genetic Epidemiology, King’s College London, London, UK

    Scott G. Wilson

  4. Medical School, University of Western Australia, Nedlands, WA, Australia

    John P. Walsh

  5. Harry Perkins Institute of Medical Research, Centre for Medical Research, QEII Medical Centre, University of Western Australia, Nedlands, WA, Australia

    Bryan K. Ward

Authors
  1. Aaron L. Magno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kassandra M. Leatherbarrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suzanne J. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Scott G. Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John P. Walsh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bryan K. Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bryan K. Ward.

Ethics declarations

Conflict of interest

The authors Aaron L Magno, Kassandra M Leatherbarrow, Suzanne J Brown, Scott G Wilson, John P Walsh and Bryan K Ward declare that they have no conflicts of interest in connection with this article. Data associated with the findings reported in this study are available from the corresponding author by request.

Human and Animal Rights

All participants gave informed consent to genetic testing which was performed as part of routine clinical care. Ethical approval for the study was granted by the Committee for Human Rights (University of Western Australia; Project C04).

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Magno, A.L., Leatherbarrow, K.M., Brown, S.J. et al. Functional Analysis of Calcium-Sensing Receptor Variants Identified in Families Provisionally Diagnosed with Familial Hypocalciuric Hypercalcaemia. Calcif Tissue Int 107, 230–239 (2020). https://doi.org/10.1007/s00223-020-00715-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-020-00715-1

Keywords

Search

Navigation