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This Article Accepted manuscript (PDF) Supplementary Data All Versions of this Article: JME-08-0164v1 42/4/331    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cole, D. Articles by Hendy, G. Search for Related Content PubMed PubMed Citation Articles by Cole, D. Articles by Hendy, G.

D Cole, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
F Yun, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
B Wong, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
A Shuen, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
R Booth, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
A Scillitani, Endocrinology Unit, San Giovanni Rotondo, Foggia, Italy
S Pidasheva, Medicine, McGill University, Montreal, Quebec, Canada
X Zhou, Medicine, McGill University, Montreal, Quebec, Canada
L Canaff, Medicine, McGill University, Montreal, Quebec, Canada
G Hendy, Physiology and Human Genetics, Dept of Medicine, Montréal, Quebec, H3A 1A1, Canada

Correspondence: Geoffrey Hendy, Email: geoffrey.hendy{at}mcgill.ca

Abstract

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 (NSHPT), 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 twelve 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 thirteenth 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 8 novel and 5 recurrent FHH mutations, along with 6 novel and 2 recurrent ADH mutations. Thus, DHPLC provides a rapid and effective means to screen for CASR mutations.