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

Purpose Autosomal dominant hypocalcemia with hypercalciuria (ADHH) is a genetic disease characterized by hypoparathyroidism with hypercalciuria. We discovered a novel activating variant (p.Tyr825Phe) of the CaSR gene in a neonate with congenital hypoparathyroidism and hypercalciuria then conducted a cell function study to prove that the CaSR variant (p.Tyr825Phe[Y825F]) is pathogenic. Methods To perform a functional study on CaSR-Y825F, we constructed expression vectors for CaSR-wild type(WT) and CaSR-Y825F. After transfection of each expression vector into HEK293 cells, we observed alterations in intracellular signaling. Then the MAP kinase signaling activity of the wild-type (CaSR-WT) and mutant type (CaSR-p.Tyr825Phe [CaSR-Y825F]) HEK293 cells was determined. Changes in intracellular calcium ions ([Ca2+]i) by extracellular calcium ion ([Ca2+]e) stimulation were quantitatively compared and analyzed. Results CaSR-Y825F showed elevated of MAP kinase signaling (pERK, pJNK, pp38) and increased [Ca2+]i levels at low [Ca2+]e stimulation. Additionally, [Ca2+]i levels in HEK293 cells with CaSR-WT and CaSR-Y825F were determined at 340 nm/380 nm wavelength ratios using Fura-2 AM. At [Ca2+]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, which were 1.04 and 1.40, respectively. Conclusion This cell function study proved that the CaSR-Y825F, which is in HEK293 cells transformed with a activating CaSR variant, elevated MAP kinase signaling(pERK, pJNK, pp38) and increased [Ca2 +] i to induce hypocalcemia.