Functional Assessment of a Constitutively Activating CASR Variant Causing Autosomal Dominant Hypocalcaemia
Bryan K Ward, Bronwyn G A Stuckey, John P Walsh, Arthur D Conigrave, Suzanne J Brown, Scott G WilsonAbstract
Context
A rare cause of hypocalcaemia, autosomal dominant hypocalcaemia type 1 (ADH1) arises from a gain-of-function variant of the calcium sensing receptor gene (CASR).
Objective
Three patients from two unrelated families, presenting with hypocalcaemia 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 three patients identified a single CASR variant which 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 receptor) was assessed by Western blot analysis and its activity measured following calcium dosing experiments using an IP-One ELISA 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 three patients. The N419S variant showed a modest increase in expression compared to wild type 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 two 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.