Related Experiment Video
Updated: Jun 26, 2026
Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
Published on: August 26, 2020
Calcium-sensing receptor mutations and denaturing high performance liquid chromatography
David E C Cole1, Francisco H J Yun, Betty Y L Wong
1Departments of Laboratory Medicine and Pathobiology, Medicine, and Genetics, University of Toronto, Ontario, Canada.
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, 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.
Insights
A new high-throughput method using denaturing high performance liquid chromatography (DHPLC) rapidly screens for calcium-sensing receptor (CASR) mutations. This technique accurately identifies mutations linked to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia (ADH).
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- The calcium-sensing receptor (CASR) regulates calcium homeostasis and is crucial in parathyroid and kidney function.
- CASR mutations cause disorders like familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalcemia (ADH).
- Accurate identification of CASR mutations is vital for managing mineral metabolism disorders.
Purpose of the Study:
- To develop and validate a high-throughput screening method for CASR mutations.
- To efficiently detect novel and recurrent mutations associated with FHH and ADH.
Main Methods:
- Utilized denaturing high performance liquid chromatography (DHPLC) for high-throughput screening of 13 CASR amplicons.
- Employed sequencing for amplicons with altered melting curves and direct sequencing for the COOH-terminal tail.
- Conducted a blinded analysis using 32 positive and 22 negative controls.
Main Results:
- Achieved 100% concordance in the blinded analysis.
- Identified eight novel and five recurrent FHH-associated mutations.
- Detected six novel and two recurrent ADH-associated mutations.
Conclusions:
- DHPLC offers a rapid and effective method for screening CASR mutations.
- This approach aids in the clinical diagnosis and management of calcium-related disorders.
- The study identified numerous new and recurring mutations in CASR.
