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Identification and Functional Characterization of Novel Calcium-Sensing Receptor Mutations in Familial Hypocalciuric Hypercalcemia and Autosomal Dominant Hypocalcemia

Departments of Medicine, Human Genetics, and Physiology, McGill University and Calcium Research Laboratory, Royal Victoria Hospital (L.D.-L., B.Y., L.C., G.N.H.), Montréal, Quebéc, Canada H3A 1A1; Department of Medicine, University of Calgary (D.A.H.), Alberta, Canada T2N 4N1; Department of Medicine, Harvard Medical School and Endocrine Unit, Massachusetts General Hospital (M.B.), Boston, Massachusetts 02114; Departments of Pediatrics and Medicine, Dalhousie University (S.R.S.), Halifax, Nova Scotia, Canada B3J 3G9; Endocrine-Hypertension Division and Membrane Biology Program, Brigham and Women’s Hospital, and Harvard Medical School (M.B., E.M.B.), Boston, Massachusetts 02114; and Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto and The Banting Institute (D.E.C.C.), Toronto, Ontario, Canada M5G 1L5

Address all correspondence and requests for reprints to: Geoffrey N. Hendy, Ph.D., Calcium Research Laboratory, Room H4.67, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebéc, Canada H3A 1A1. E-mail: . gnhendy{at}med.mcgill.ca

Abstract

Familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcemia (ADH), in which calcium homeostasis is disordered, are associated with mutations in the calcium-sensing receptor (CASR). Six unrelated kindreds with FHH and/or NSHPT and two unrelated kindreds with ADH were studied. Direct sequence analysis of the exons of the CASR gene identified heterozygous mutations in six of the kindreds with FHH and in one of those with ADH. We performed functional analyses on the novel missense and insertion/frameshift mutants by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. All mutant receptors were expressed at a similar level to that of the wild type; however, whereas mutants R220W and A835T (the ADH mutant) were fully glycosylated and were visualized on the cell surface, glycosylation of mutants G549R and C850^851 ins/fs was impaired, resulting in reduced cell surface staining. In fura-2-loaded HEK293 cells expressing the wild-type or mutant receptors, the inactivating R220W mutant produced a significant shift to the right relative to the wild-type CASR in the cytosolic calcium response to increasing extracellular calcium concentrations and the G549R and C850^851 ins/fs mutants were without detectable activity. The activating A835T mutation resulted in a shift to the left in the cytosolic calcium response to extracellular calcium concentrations relative to the wild type. Our studies have identified novel CASR mutations that alter the function of the CASR in several different ways.

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