Functional assessment of a constitutively activating CASR variant causing autosomal dominant hypocalcemia

Abstract Context A rare cause of hypocalcemia, autosomal dominant hypocalcemia type 1 (ADH1) arises from a gain-of-function variant of the calcium-sensing receptor gene (CASR). Objective Three patients from 2 unrelated families, presenting with hypocalcemia and other biochemical parameters consistent with ADH1, were examined for variants in the CASR with the aim to functionally assess any variant detected to confirm the ADH1 diagnosis. Methods Sanger sequencing of the coding region of the CASR from the 3 patients identified a single CASR variant that was generated by site-directed-mutagenesis in the CASR as a FLAG-tagged construct in the mammalian expression vector pcDNA3.1. The variant's expression in HEK293 cells (compared to FLAG-tagged wild-type [WT] receptor) was assessed by Western blot analysis and its activity measured following calcium dosing experiments using an IP-One enzyme-linked immunosorbent assay. Results Sequence analysis revealed the presence of a heterozygous missense variant in the CASR, an adenine to guanine transition at nucleotide 1256 causing an asparagine to serine substitution at amino acid 419 (N419S) in the CaSR’s Venus flytrap domain in all 3 patients. The N419S variant showed a modest increase in expression compared to the WT receptor. Significantly, the IP-One assay demonstrated that the variant is constitutively active in the absence of Ca++ ions and that this gain-of-function is maintained at physiologically relevant Ca++ ion concentrations. Conclusion The N419S CASR variant affecting 2 separate families is constitutively activating and therefore causative of ADH1. This is the first report of a constitutively active variant affecting the extracellular domain of the CaSR.