Objectives: The calcium-sensing receptor (CaSR) is a G-protein coupled receptor that regulates the secretion of the parathyroid hormone (PTH) in response to extracellular calcium ([Ca2+]o) as well as the re-absorption of urinary calcium. Inherited abnormalities in the CaSR gene cause either hyper- or hypocalcemia. The familial hypocalciuric hypercalcemia (FHH) is characterized by modest elevation of the S-Ca, relative hypocalciuria and PTH that is inappropriately normal or slightly increased. Patients with autosomal dominant hypocalcemia (ADH) have low S-Ca, relative hypercalciuria with inappropriately low PTH.
We present six novel CaSR mutations (C568Y, W718X, M734R, L849P, E767Q, A844T). The first four were found in patients who clinically presented with possible FHH (S-Ca 2.74–2.88 mmol/l), whereas the other two were detected in patients suffering from possible ADH (S-Ca 1.85 and 1.98 mmol/l).
Methods: Mutant cDNAs were generated and transfected into HEK293 cells. Functional characterization was performed by measuring changes in intracellular calcium in response to varying [Ca2+]o.
Results: The wild type (wt)-receptor had an EC50 for [Ca2+]o of 4.9±0.4 mM (n=7). The putative inactivating mutations were unresponsive to [Ca2+]o up to concentrations of 30 mM. In order to mimic the heterozygous state cells were cotransfected with mutant and wt-CaSR. This caused a right-shift of the dose-response curves to [Ca2+]o compared to the wt-CaSR (C568Y/wt: 8.3 mM, W718X/wt: 6.8 mM, M734R/wt: 6.9 mM, and L849P/wt: 7.8 mM). In HEK293 cells transfected with the putative activating mutations a left-shift in the dose-response curves to [Ca2+]o were observed (E767Q: 2.9 mM and A844T: 3.45 mM).
Conclusions: In patients with unclear or borderline biochemical parameters the molecular genetic analysis of the CaSR is important to differentiate between FHH and primary hyperparathyroidism and between ADH and idiopathic hypoparathyroidism. Functional analysis of mutations found in these patients finally proves the link between the mutation and the clinical presentation.
