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

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Familial hypocalciuric hypercalcaemia: a review.

PURPOSE OF REVIEW: Hypercalcaemia is a potentially life-threatening condition. Familial hypocalciuric hypercalcaemia (FHH) is a rare, lifelong, benign condition. It is important to separate this condition from other hypercalcaemic states such as hypercalcaemia of malignancy and primary hyperparathyroidism (PHPT). RECENT FINDINGS: FHH is caused by inactivating mutations in the calcium sensing receptor (CASR) gene leading to a general calcium-hyposensitivity, compensatory hypercalcaemia and hypocalciuria. The inheritance of FHH is autosomal dominant. Similar to PHPT, FHH is characterized by hypercalcaemia, unsuppressed or elevated plasma parathyroid hormone, and typically normal renal function. The phenotype is normal, and hypercalcaemic symptoms are generally absent. The hallmark is a relatively low urine calcium excretion in contrast to PHPT, in which urine calcium excretion is increased. The vitamin D status as measured by plasma 25-hydroxyvitamin D has been reported to be normal with normal seasonal variations, whereas plasma 1,25-dihydroxyvitamin D has been found slightly increased compared to normal. Bone mineral density Z-scores are normal in spite of a slightly increased bone turnover. Differential diagnoses include mainly PHPT, but in some cases also hypercalcaemia of malignancy and use of thiazide diuretics. SUMMARY: In general, FHH does not require treatment. We recommend a two-step diagnostic procedure. First, the calcium/creatinine clearance ratio is measured from a 24-h urine. Second, all patients with calcium/creatinine clearance ratio of 0.020 or less are tested for mutations in the CASR gene. The diagnostic sensitivity of this setup is 98%.

Multiplex ligation-dependent probe amplification (MLPA) screening for exon copy number variation in the calcium sensing receptor gene: no large rearrangements identified in patients with calcium metabolic disorders.

BACKGROUND: Mutation screening of the CASR by DNA sequencing is commonly used in the diagnosis of disorders of calcium metabolism, such as familial hypocalciuric hypercalcaemia (FHH). Exon copy number variation is not detected by currently used molecular genetic screening methods, and might be a genetic cause of inherited forms of hyper- or hypocalcaemia caused by the CASR. OBJECTIVE: We wanted to further evaluate possible genetic causes for disorders of calcium metabolism, by investigating the prevalence of exon copy number variations, such as large deletions or duplications of the CASR. PATIENTS AND METHODS: The study included 257 patient samples referred to our laboratory for molecular genetic analysis of the CASR gene. A total of 245 were patients suspected to have FHH, while the remaining 12 samples represent patients with a phenotype of idiopathic hypocalcaemia/hypoparathyroidism. All samples were previously found negative for CASR mutations. Multiplex ligation-dependent probe amplification was used to screen the patients for exon copy number variations. RESULTS: All exons were amplified with mean normalised ratios between 0.98 and 1.06. We did not identify any exon copy number variation in the CASR. Bioinformatic analyses revealed that the CASR gene contains 52% repeated elements, of which approximately 6% consist of Alu elements. CONCLUSIONS: The present study indicates that including CASR MLPA analysis as a routine part of the diagnostic setup is not necessary, but could still be of interest in cases with a clear family history and no evidence of missense mutations in the CASR gene.

Molecular genetic analysis of the calcium sensing receptor gene in patients clinically suspected to have familial hypocalciuric hypercalcemia: phenotypic variation and mutation spectrum in a Danish population.

CONTEXT: The autosomal dominantly inherited condition familial hypocalciuric hypercalcemia (FHH) is characterized by elevated plasma calcium levels, relative or absolute hypocalciuria, and normal to moderately elevated plasma PTH. The condition is difficult to distinguish clinically from primary hyperparathyroidism and is caused by inactivating mutations in the calcium sensing receptor (CASR) gene. OBJECTIVE: We sought to define the mutation spectrum of the CASR gene in a Danish FHH population and to establish genotype-phenotype relationships regarding the different mutations. DESIGN AND PARTICIPANTS: A total of 213 subjects clinically suspected to have FHH, and 121 subjects enrolled as part of a family-screening program were studied. Genotype-phenotype relationships were established in 66 mutation-positive index patients and family members. MAIN OUTCOME MEASURES: We determined CASR gene mutations, and correlating levels of plasma calcium (albumin corrected), ionized calcium (pH 7.4), and PTH were measured. RESULTS: We identified 22 different mutations in 39 FHH families. We evaluated data on circulating calcium and PTH for 11 different mutations, representing a spectrum of clinical phenotypes, ranging from calcium concentrations moderately above the upper reference limit, to calcium levels more than 20% above the upper reference limit. Furthermore, the mean plasma PTH concentration was within the normal range in eight of 11 studied mutations, but mild to moderately elevated in families with the mutations p.C582Y, p.C582F, and p.G553R. CONCLUSIONS: The present data add 19 novel mutations to the catalog of inactivating CASR mutations and illustrate a variety of biochemical phenotypes in patients with the molecular genetic diagnosis FHH.