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

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+16T/C, c.2956G>T and c.2968A>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 (PolyPhen2 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. ► Variant scanning by HRM analysis detects 96% of rare CASR gene variants. ► HRM scanning and genotyping frequent CASR variants provide a simple new workflow. ► 24 novel rare CASR variants were detected using high-resolution melting analysis.