A cell function study on calcium regulation of a novel calcium-sensing receptor mutation (p.Tyr825Phe)

PURPOSE: Autosomal dominant hypocalcemia with hypercalciuria is a genetic disease characterized by hypoparathyroidism with hypercalciuria. We discovered a novel variant (p.Tyr825Phe[Y825F]) of the <i>CASR</i> gene in a neonate with congenital hypoparathyroidism and hypercalciuria and conducted a cell function study to determine whether the CASR-Y825F variant was pathogenic. METHODS: To perform a functional study on CaSR-Y825F, we constructed expression vectors expressing wild-type (WT) CASR and CASR-Y825F. After transfection of each expression vector into HEK293 cells, we examined alterations in intracellular signaling. Mitogen-activated protein kinase (MAPK) signaling activity of HEK293 cells expressing CASR-WT or CASR-Y825F was determined. Changes in intracellular calcium ions ([Ca(2+)]i) by extracellular calcium ion ([Ca(2+)]e) stimulation were quantitatively compared and analyzed. RESULTS: Cells expressing CASR-Y825F showed elevated of MAPK signaling (phospho-ERK [pERK], phospho-JNK [pJNK], phospho-p38 [pp38]) and increased [Ca(2+)]i levels at low [Ca(2+)]e stimulation compared with cells expressing CASR-WT. Additionally, [Ca(2+)]i levels in HEK293 cells expression CASR-WT and CASR-Y825F were determined at 340 nm/380 nm wavelength ratios using Fura-2 AM. At [Ca(2+)]e concentrations of 2.5 mM and 3 mM, the ratios of CASR-Y825F cells were higher (2.6 and 3.5, respectively) than those of CASR-WT cells (1.04 and 1.40, respectively). CONCLUSIONS: This cell function study proved that the CASR-Y825F expressed in HEK293 cells elevated MAPK signaling (pERK, pJNK, pp38) and increased [Ca(2+)]i to induce hypocalcemia.