A family of autosomal dominant hypocalcemia with a positive correlation between serum calcium and magnesium: identification of a novel gain of function mutation (Ser(820)Phe) in the calcium-sensing receptor.

To date about 20 activating mutations in the calcium-sensing receptor (CaR) gene have been identified to cause autosomal dominant hypocalcemia (ADH) or sporadic hypoparathyroidism. We report a novel activating mutation in the CaR gene in a Japanese family with ADH. The proband, a 15-yr-old boy, and 5 other patients in 3 generations were asymptomatic, except for the proband's grandmother who had a history of seizures. They showed mild hypocalcemia (1.68-1.98 mmol/liter) with normal urinary calcium excretion and low normal serum PTH levels. Their serum magnesium concentrations were below normal in 3 adults and within the normal range in 3 teenagers. There was a significant positive correlation (r = 0.90; P < 0.05) between the serum calcium and magnesium concentrations of 6 affected members. Nucleotide sequencing revealed that the proband had a known polymorphism (Gly(990)Arg) and a novel heterozygous mutation substituting phenylalanine for serine at codon 820 (Ser(820)Phe) in the sixth transmembrane helix of the CaR. In other family members, the Ser(820)Phe mutation cosegregated with hypocalcemia. The mutation was not detected in 50 control subjects. The Gly(990)Arg polymorphism was observed in 8 of 9 family members with or without hypocalcemia and in 36 of 50 controls. The sensitivity of the Ser(820)Phe mutant CaR to calcium was assessed using transiently transfected HEK293 cells and measuring the increases in intracellular Ca(2+) concentrations in response to the changes in extracellular Ca(2+). The concentration-response curve of the mutant receptor was left-shifted, and its EC(50) (2.5 mM) was significantly (P < 0.05) lower than that of the wild-type CaR (3.3 mM). We conclude that the Ser(820)Phe mutation in the CaR caused ADH in this family. The positive correlation between serum calcium and magnesium levels observed in this family may support the concept that renal CaR acts as a magnesium sensor as well as a calcium sensor.

Involvement of cyclo-oxygenase-2 in osteoclast formation and bone destruction in bone metastasis of mammary carcinoma cell lines.

We previously reported that mouse mammary carcinoma cell lines (MMT060562 and BALB/c-MC) induced osteoclast formation through production of prostaglandin E2 (PGE2) in cocultures with mouse bone marrow cells, but the mechanism(s) of PG production remained unclear. In the present in vitro and in vivo studies, we tested the involvement of cyclo-oxygenase-2 (COX-2), an inducible rate-limiting enzyme in PG biosynthesis, in the stimulation of osteoclast formation by mouse mammary carcinoma cell lines. Addition of a selective COX-2 inhibitor, JTE-522, to cocultures of mammary carcinoma cell lines and bone marrow cells lowered PGE2 concentration in the culture media and inhibited osteoclast formation in a dose-dependent manner. Northern blotting showed a very high level of COX-2 messenger RNA (mRNA) expression in MMT060562. The mRNA expression was low in BALB/c-MC, but it increased when BALB/c-MC and bone marrow cells were cocultured. The results of immunocytochemistry for COX-2 protein in respective cultures were compatible with the results of COX-2 mRNA. In vivo, BALB/c-MC injected into the heart of Balb/c mice metastasized to bone and formed osteolytic lesions in their hindlimbs. Histological examination revealed that tumor cells had metastasized to the bone marrow cavity and destroyed the bone trabeculae. Immunohistochemistry demonstrated that bone marrow stromal cells adjacent to tumor cells expressed COX-2 protein. These findings suggest that COX-2 plays an important role in the osteolysis of bone metastasis in vivo as well as in osteoclast formation in cocultures used as an in vitro model of metastatic bone disease.