SFEBES2023 Oral Communications Bone and Calcium (6 abstracts)
A large in-frame deletion of the calcium-sensing receptor extracellular domain causes familial hypocalciuric hypercalcaemia type 1 (FHH1) and is partially responsive to cinacalcet
Kreepa Kooblall 1 , Fadil Hannan 2 , Charlotte van Waes 1 , Mark Stevenson 1 , Kate Lines 1 , David Evans 3 , Catherine Moorwood 3 , Martina Owens 3 , Antoinette Tuthill 4 & Rajesh Thakker 1
1Academic Endocrine Unit, Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Churchill Hospital, Headington, Oxford, United Kingdom. 2Nuffield Department of Women’s and Reproductive Health, University of Oxford, Oxford, United Kingdom. 3Exeter Genomics Laboratory, Royal Devon University Healthcare NHS Foundation Trust, Exeter, United Kingdom. 4Cork University Hospital, Wilton, Ireland
Familial hypocalciuric hypercalcaemia type 1 (FHH1) is mainly caused by loss-of-function missense mutations of the extracellular calcium-sensing receptor (CaSR), which is a parathyroid- and kidney-expressed G-protein coupled receptor that plays a pivotal role in mineral metabolism. Here, we report the unusual occurrence of a novel heterozygous in-frame CASR exon 4 deletion, c.(492+1_493-1)_(1377+1_1378-1)del, in a family with FHH1. This mutation is predicted to delete 295 amino acids from the Ca2+-binding region of the CaSR extracellular domain (ECD), resulting in loss of >25% of the CaSR protein. We hypothesised that this large in-frame deletion would affect CaSR signalling, expression, and responsiveness to cinacalcet, which is a CaSR-positive allosteric modulator. In vitro expression studies involving transient transfection of wild-type (WT) and mutant N-terminal-FLAG tagged CaSR constructs into HEK293 cells, followed by immunofluorescence analysis showed that the mutant CaSR protein localised to the cell surface. However, Western blot analysis demonstrated that the mutant CaSR protein was 3.7-fold less abundant (P<0.01) and truncated (~30% smaller), compared to WT CaSR. Moreover, dose-dependent fluo-4 intracellular calcium mobilisation assays showed that, compared to WT CaSR, the mutant protein abolished Ca2+i-dependent receptor signalling, while HEK293 cells co-transfected with WT and mutant CaSR constructs to mimic the heterozygous state showed a significant rightward shift in the Ca2+i-dependent receptor response curve (P<0.0001). HEK293 cells expressing only the mutant CaSR were unresponsive to treatment with 100nM cinacalcet. However, cinacalcet treatment normalised Ca2+i-dependent receptor responses in HEK293 cells expressing a 1:1 ratio of WT to mutant CaSR. These findings indicate that cinacalcet has therapeutic potential for symptomatic FHH1 patients harbouring the heterozygous form of this mutation. In summary, this study has identified, functionally characterised, and evaluated targeted therapy for the first known naturally occurring deletion of the CaSR Ca2+-binding region in a family with FHH1.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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