Endocrine Abstracts

Mutational analysis of the calcium-sensing receptor (CaSR) is frequently undertaken to confirm a diagnosis of familial hypocalciuric hypercalcaemia (FHH) and autosomal dominant hypocalcaemic hypercalciuria (ADHH). However, functional characterization of these CaSR mutations to demonstrate loss-of-function for FHH mutations and gain-of-function for ADHH mutations is infrequently performed. We demonstrate the importance of pursuing in vitro studies that investigate the functional consequences of such CaSR mutants, by revealing that a previously reported CaSR mutant (Glu250Lys), which involves substitution of a negatively charged, evolutionary conserved, glutamate residue for a positively charged lysine residue, in probands with FHH and primary hyperparathyroidism (1,2) is not a mutation, but instead a functionally neutral polymorphism. Our recent finding of the occurrence of this Glu250Lys alteration in a proband with ADHH raised difficulties in providing a plausible explanation for this possible CaSR mutation causing two opposingly different phenotypes. We therefore pursued functional characterisation studies of this previously reported CaSR mutant (1,2) by transient transfection of the wild-type (Glu250) and purported mutant Lys250 CaSRs in human embryonic kidney 293 cells. This revealed the concentration-response curve and EC50 of the Lys250 CaSR variant to not significantly differ (P= NS) from that of the wild-type receptor (Lys250 versus wild-type = 2.56 ± 0.02 mM versus 2.49 ± 0.06 mM). Moreover, use of a bioinformatics program (Polyphen) predicted the Lys250 CaSR to be a benign variant and examination of data derived from The Exome Project revealed the occurrence of this Lys250 CaSR in 0.6% of normal, unrelated individuals, consistent with the Glu250Lys missense substitution representing a functionally neutral polymorphism. Our findings emphasise the value of undertaking in vitro functional characterisation of CaSR variants. References: 1. Nissen PH et al. (2007) J Clin Endocrinol Metab 92: 4373–4379. 2. Simonds WF et al. (2002) Medicine (Baltimore) 81: 1–26.

Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.