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Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto, Ontario, Canada M5G 1L5¹ Endocrinology Unit, Hospital Casa Sollievo della Sofferenza, Istituto di Ricovero e Cura a Carrattere Scientifico, San Giovanni Rotondo (Foggia), 71013 Italy² Hormones and Cancer Research Unit, Calcium Research Laboratory, Departments of Medicine, Physiology, and Human Genetics, Royal Victoria Hospital, McGill University, Room H4.67, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1

(Correspondence should be addressed to G N Hendy; Email: geoffrey.hendy{at}mcgill.ca)

The calcium-sensing receptor (CASR), a plasma membrane G-protein-coupled receptor, is expressed in parathyroid gland and kidney, and controls systemic calcium homeostasis. Inactivating CASR mutations are associated with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism, and activating mutations cause autosomal dominant hypocalcemia (ADH). CASR mutation identification plays an important role in the clinical management of mineral metabolism disorders. We describe here a high-throughput method using screening with denaturing high performance liquid chromatography (DHPLC) to initially interrogate 12 amplicons covering translated exons and exon/intron boundaries, followed by sequencing of any amplicon with a modified melting curve relative to wild type, and direct sequencing of a 13th amplicon encoding the COOH-terminal tail to distinguish causative mutations from three common missense single nucleotide polymorphisms. A blinded analysis of 32 positive controls representing mutations throughout the CASR sequence, as well as 22 negative controls, yielded a concordance rate of 100%. We report eight novel and five recurrent FHH mutations, along with six novel and two recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.