Identification of rare and frequent variants of the CASR gene by high-resolution melting.

[familial hypocalciuric hypercalcemia]

Calcium metabolic disorders like familial hypocalciuric hypercalcemia (FHH ) and autosomal dominant familial isolated hypoparathyroidism (FIH ) can be caused by rare variants of the calcium sensing receptor gene (CASR ). Molecular genetic screening of the CASR is often based on DNA sequencing .We sought to develop a pre-screening method in the diagnostic procedure and pursued variant scanning by high -resolution melting analysis (HRM ) on a LightScanner instrument . We used 50 samples , representing 45 different rare variants , to validate the HRM method . In addition , we implemented small amplicon genotyping of three frequent CASR variants (c .1732 +16 T /C , c .2956 G >T and c .2968 A >G ).Using HRM , we identified 43 of 45 variants confidently (~96 %) while two variants escaped immediate detection . Implementing this method in clinical use further resulted in the identification of seven new CASR variants and nine recurrent . HRM variant scanning , in combination with small amplicon genotyping , provides a simple workflow with reduced sequencing burden . Bioinformatics analyses using two freely available prediction tools (PolyPhen 2 and SIFT ) for evaluating amino acid substitutions were compared and indicated discrepancies in the prediction for 25 % of the variants .This study demonstrates the utility of HRM as a pre-screening method , adds 24 novel rare CASR variants , and further emphasizes the importance of clinical decision making based on all available information rather than bioinformatics alone .