Identification and characterization of D410E, a novel mutation in the loop 3 domain of CASR, in autosomal dominant hypocalcemia and a therapeutic approach using a novel calcilytic, AXT914

Background: Autosomal dominant hypocalcemia (ADH) is a rare disorder characterized by benign hypocalcemia, inappropriately low parathyroid hormone (PTH) levels and mostly hypercalciuria. ADH can be caused by activating mutations of the calcium-sensing receptor (CaSR) gene located on chromosome 3q13.3-21. Calcium-sensing receptor plays a pivotal role in the regulation of calcium homeostasis and is abundantly expressed in parathyroid gland, thyroid C cell, and renal tubular system. Activation of CaSR by increased Ca2+ inhibits PTH secretion, stimulates calcitonin secretion, and promotes urinary Ca2+ excretion, and thereby maintains the Ca2+ at the normal level. Herein, we report a novel activating mutation in the CaSR gene in a Korean family with ADH. Meanwhile, antagonist of the CaSR, calcilytics could be a therapeutic option in the treatment of ADH. Method: We identified a 55-yr-old woman with mild hypocalcemia (7.7 mg/dL) and hypercalciuria (24-hr urine Ca: 868 mg/d) caused by missense mutations of the CaSR gene. She showed low normal serum PTH level (10.14 pg/mL). We performed mutational analysis of the genes encoding GCMB, pre-pro-PTH and CaSR using PCR-amplified genomic DNA in her family members. The ability of wild-type and mutant CaSR to activate the MAPK signaling cascade was assessed by examining phosphorylation of ERK1/2. Intacellular Ca2+ concentration was measured by Fura-2 dye. Blocking of CaSR with calcilytics, AXT914 was also tested by Fura-2 with a variety of concentrations. Result: Direct sequencing analysis of the CaSR gene showed that the proband and her daughter possess a T to A transition at nucleotide 1230 resulting in a D410E missense mutation in exon 4 of the CaSR. No mutation was detected in GCMB and Prepro-PTH genes. HEK293 cells were stably transfected with plasmids encoding wild-type or mutant CaSR genes. Escalation of the extracellular Ca2+ concentration from 0.5 to 5.0 mM resulted in increased phosphorylation of ERK1/2 and escalation of the extracellular Ca2+ concentration from 1.0 to 10 mM resulted in increased intracellular Ca2+ detected by Fura-2 in mutant CaSR-expressing cell than wild-type-expressing one. These results indicate that D410E mutation of CaSR is associated with ADH in this family. Finally, AXT914 successively blunted the increased intracellular signaling via CaSR. Conclusion: Over 60 activating mutations in the CaSR gene have been identified to cause ADH so far. Here we add one more activating mutation that causes ADH. This could be of interest because this novel mutation occurred in the loop 3 region of the VFT domain in CaSR where little was known to be important in its function. Further clinical study is needed to validate the effectiveness of calcilytics in the treatment of ADH in vivo.