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

Objective Activating mutations of the calcium-sensing receptor (CASR) gene are associated with autosomal dominant hypocalcemia (ADH) characterized by benign hypocalcemia, inappropriately low (PTH) levels and mostly hypercalciuria. Herein, we report a novel activating mutation in the CASR gene in a Korean family with ADH. Method The CASR gene was sequenced in the patient with ADH. The identified mutations were also evaluated in the patient's family members by PCR-based sequencing. For functional studies, we examined phosphorylation of ERK1/2. In addition, intracellular Ca2+ mobilization and the effects of the calcilytic, AXT914 were measured using fluorophore Fura-2 dye. Result Direct sequencing analysis of the CASR gene showed that the proband and her daughter possess a novel mutation c.1230T>A, resulting in a D410E missense mutation on exon 4 of the CASR gene. Escalation of the extracellular Ca2+ concentration resulted in stronger phosphorylation of ERK1/2 and higher levels of intracellular Ca2+ in HEK293 cells expressing mutant CASR, compared with wild-type CASR. The increase in intracellular Ca2+ signalling via CASR was successively blunted by treatment with AXT914. Conclusions 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. The novel activating mutation (D410E) occurred in the loop 3 region of CASR, where its function was believed to be of little importance; therefore, this mutation may be of interest. Further clinical study will be needed to validate the effectiveness of calcilytics in treatment of ADH in vivo.