Novel mutations in the calcium-sensing receptor gene associated with biochemical and functional differences in familial hypocalciuric hypercalcaemia

• Published in: Clinical endocrinology (Oxford) Vol. 64; no. 5; pp. 580 - 587
• Main Authors: Ward, Bryan K., Magno, Aaron L., Blitvich, Bradley J., Rea, Alexander J., Stuckey, Bronwyn G. A., Walsh, John P., Ratajczak, Thomas
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.05.2006; Blackwell; Wiley Subscription Services, Inc
• Subjects: Animals; Biological and medical sciences; Biological Assay; Calcium - metabolism; Calcium - pharmacology; Calcium - urine; Cell Line; DNA Mutational Analysis; Endocrinopathies; Fundamental and applied biological sciences. Psychology; Heterozygote; Humans; Hypercalcemia - genetics; Hypercalcemia - metabolism; Least-Squares Analysis; Medical sciences; Mutagenesis, Site-Directed; Mutation, Missense; Receptors, Calcium-Sensing - analysis; Receptors, Calcium-Sensing - genetics; Receptors, Calcium-Sensing - metabolism; Transfection - methods; Vertebrates: endocrinology; Hypercalcemia; Metabolic disorder; Hydroelectrolytic balance disorder; Calcium; Gene; Hypocalciuria; Genetics; Biochemistry; Mutation; Calcium-sensing receptor; Inorganic element; Endocrinology
• ISSN: 0300-0664; 1365-2265
• DOI: 10.1111/j.1365-2265.2006.02512.x

Summary Objective  Heterozygous inactivating mutations of the calcium‐sensing receptor (CaR) gene cause familial hypocalciuric hypercalcaemia (FHH), a generally benign disorder characterized by mild to moderate PTH‐dependent hypercalcaemia. We aimed to identify the causative CaR mutations in three families with FHH and examine the correlation between type of mutation and biochemical and/or functional phenotypes. Patients, design and measurements  The CaR gene from FHH family members was assessed for mutations by direct DNA sequencing and mutations were confirmed by restriction enzyme analysis. Functional studies on two missense mutations were conducted by introducing them by site‐directed mutagenesis into the CaR cloned into a mammalian expression vector, and assessing calcium responsiveness using an inositol phosphate (IP) assay in HEK293 cells. Biochemical data from patients heterozygous for each type of mutant were correlated with functionality. Results  Two novel nonsense mutations (R25stop and K323stop) and one novel missense mutation (G778D) were identified. The G778D mutant receptor and another mutation identified in an earlier study (L174R) demonstrated a complete lack of Ca2+ responsiveness using the IP assay. When cotransfected with wild‐type receptor, the mutant receptors demonstrated a dominant‐negative effect on wild‐type receptor response, with L174R having a more pronounced effect than G778D. Significantly more severe hypercalcaemia and a trend towards higher PTH levels were observed in patients heterozygous for CaR mutants with a stronger dominant‐negative effect. Conclusions  Naturally occurring CaR mutations with differences in dominant‐negative effect on wild‐type receptor demonstrate differences in biochemical severity in FHH.