Hypercalcemic and Hypocalcemic Disorders via the CASR Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1457 CASR$870.00 81405 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

CASR mutations are detected in around 65% of FHH patients (Hannan et al. Best Pract Res Clin Endocrinol Metab 27(3):359-371, 2013). In a cohort of 294 patients with clinical features suggestive of hypercalcemic (FHH and NSHPT) or hypocalcemic (ADH) disorders, the overall CASR mutation detection rate is 29% (26% in FHH and NSHPT; 41% in ADH) (Hannan et al. Hum Mol Genet 21(12):2768-2778, 2012). CASR mutation detection rate in a large cohort of patients with PTPH or Bartter syndrome type V is unknown because these CASR mutations have been only reported in limited individual cases.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 CASR$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Defects of the calcium-sensing receptor (CaSR) can cause both hypercalcemic and hypocalcemic disorders (Hannan et al. Best Pract Res Clin Endocrinol Metab 27(3):359-71, 2013; Egbuna et al. Best Pract Res Clin Rheumatol 22(1):129-48, 2008). Loss-of-function (inactivating) mutations of its encoding gene CASR lead to three hypercalcemic disorders: familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT) and primary hyperparathyroidism (PHPT). On the other hand, gain-of-function (activating) CASR mutations have been associated with two hypocalcemic disorders: autosomal dominant hypocalcemia (ADH) and Bartter syndrome type V. 

Familial hypocalciuric hypercalcemia (FHH) is a heritable disorder of mineral homeostasis characterized by lifelong elevation of serum calcium concentrations. FHH patients are usually asymptomatic, and the disorder is generally considered benign. Clinical features of FHH include hypermagnesemia and low urinary calcium excretion. FHH patients have normal or mildly elevated circulating parathyroid hormone (PTH) levels. In uncommon symptomatic cases, adult patients have chondrocalcinosis and pancreatitis while some children may develop neonatal severe hyperparathyroidism (NSHPT). FHH is a genetically heterogeneous disorder and consists of three variants:  FHH1, FHH2 and FHH3. FHH1 (OMIM# 145980) is caused by dominant loss-of-function CASR mutations. The age of FHH onset is mostly in infancy but severe FHH can present in either childhood or early adulthood.

Neonatal severe hyperparathyroidism (NSHPT; OMIM# 239200) is a life-threatening disorder characterized by severe hypercalcaemia, hyperparathyroidism, enlarged parathyroid glands, hypotonia, bone demineralization, fractures, ribcage deformities, respiratory distress and failure to thrive. NSHPT usually manifests in the first 6 months of life. Early diagnosis of NSHPT is critical because usually a parathyroidectomy is required. Severity of disease manifestations can vary within the same family.

Within a variable phenotypic spectrum of hypercalcemia associated with loss-of-function CASR mutations, primary hyperparathyroidism (PHPT) is comparable to FHH with overlapping features but represents a distinct clinical entity. The age of PHPT onset is in adulthood. The severity of hypercalcemia in PHPT patients is variable. In addition to hypercalcemia, PHPT patients may have non-suppressed urinary calcium excretion, elevated serum PTH concentrations, kidney stones, and/or osteoporosis. Parathyroid adenomas or hyperplasia are also common.    

Autosomal dominant hypocalcemia (ADH; OMIM# 601198) is characterized by mild to moderate hypocalcemia. ADH patients are generally asymptomatic, but some patients may have neuromuscular symptoms such as paresthesiae, carpopedal spasm and seizures. Other common features of ADH include hyperphosphatemia, hypomagnesemia and relative hypercalciuria.

Bartter syndrome is a clinically and genetically heterogeneous group of electrolyte disorders characterized by renal salt wasting, which results in volume depletion with consequent hyperreninemic hyperaldosteronism and hypokalemic alkalosis. Bartter syndrome type V is caused by activating CASR mutations.


CASR has 6 coding exons that encode the calcium-sensing receptor, a G-protein-coupled receptor (GPCR), which is essential in extracellular calcium homeostasis and regulation of salt-water metabolism (Pollak et al. Cell 75(7):1297-303, 1993; Hannan et al., 2013). Genetic defects located throughout the CASR gene include missense, nonsense, splicing site mutations, and small deletion/insertions, while large deletions and insertions are very rare (Human Gene Mutation Database). The majority (>50%) of mutations associated with hypercalcemic and hypocalcemic disorders are located in the extracellular domain (ECD) of CaSR (Hannan et al. Hum Mol Genet 21(12):2768-2778, 2012).

Familial hypocalciuric hypercalcemia type 1 (FHH1) is inherited in an autosomal dominant manner and is caused by heterozygous CASR mutations (Pollak et al., 1993). FHH-associated CASR mutations account for approximately 50% of all documented CASR mutations (Human Gene Mutation Database; Hannan et al., 2013). These FHH-associated mutations are predominantly missense substitutions but other type mutations have also been reported. Compared to missense substitutions, truncating mutations are associated with a milder hypercalcemic phenotype, suggesting a dominant-negative effect of missense substitutions. Notably, the majority of FHH-associated mutations are located within the extracellular domain (ECD) of CaSR and clustered at negatively charged regions. CASR mutations are detected in only around 65% of FHH patients; the other two recently identified FHH genes are AP2S1 (FHH3) and GNA11 (FHH2) (Nesbit et al. Nat Genet 45(1):93-97, 2013; Nesbit et al. N Engl J Med 368(26):2476-2486, 2013).

Neonatal severe hyperparathyroidism (NSHPT) is an autosomal recessive disorder caused by familial homozygous or compound heterozygous loss-of-function CASR mutations (Pollak et al., 1993). Interestingly, dominant-acting heterozygous CASR mutations have also been reported to arise de novo in sporadic NSHPT patients with milder phenotypes (Pearce et al. J Clin Invest 96(6):2683-2692, 1995; Bai et al. J Clin Invest 99(1):88-96, 1997). Approximately 10% of documented CASR mutations are associated with NSHPT, more than 40% of which are truncating mutations (either nonsense or frameshift) (Human Gene Mutation Database; Hannan et al., 2013).

Primary hyperparathyroidism (PHPT) is caused by either heterozygous or homozygous loss-of-function CASR mutations (Hannan et al. Clin Endocrinol. 73(6):715-22, 2010; Hannan et al., 2013). Hypercalcemic disorders after infancy, including PHPT and severe FHH, may be associated with the degree of function loss resulting from underlying CASR mutations. Unknown genetic modifiers could also contribute to the variability of these hypercalcemic phenotypes.

Autosomal dominant hypocalcemia (ADH) is caused by gain-of-function CASR mutations (Pollak et al. Nat Genet 8(3):303-307, 1994). Approximately 30% of documented CASR mutations are associated with ADH, 95% of which are missense mutations (Hannan et al., 2013). There are two hotspots of ADH-associated CASR mutations: the second peptide loop of the ECD (residues 116–136), and a region that encompasses transmembrane domains 6 and 7 and the intervening third extracellular loop (residues 819–837). The other recently identified ADH gene is GNA11 (Nesbit et al., 2013).

Bartter syndrome type V is an autosomal dominant disorder caused by activating gain-of-function CASR mutations (Vargas-Poussou et al. J Am Soc Nephrol 13(9):2259-2266, 2002; Watanabe et al. Lancet 360(9334):692-694, 2002; Vezzoli et al. J Nephrol 19(4):525-528, 2006). To date, only four CASR mutations (K29E, L125P, C131W and A843E) have been found in Bartter syndrome type V.

Testing Strategy

This test involves bidirectional Sanger DNA sequencing of all 6 coding exons of CASR.  The entire coding region and ~20 bp of flanking non-coding DNA on either side of each splice site are sequenced. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Candidates for this test are patients with any of the hypercalcemic or hypocalcemic disorders listed. Testing is also indicated for family members of patients who have known CASR mutations.


Official Gene Symbol OMIM ID
CASR 601199
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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Genetic Counselors
  • Bai, M. et al. (1997). "In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia." J Clin Invest 99(1):88-96. PubMed ID: 9011580
  • Egbuna, O. et al. (2008). "Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations." Best Pract Res Clin Rheumatol 22(1):129-148. PubMed ID: 18328986
  • Hannan, F. et al. (2010). “A homozygous inactivating calcium-sensing receptor mutation, Pro339Thr, is associated with isolated primary hyperparathyroidism: correlation between location of mutations and severity of hypercalcaemia.” Clin Endocrinol 73(6):715-722. PubMed ID: 20846291
  • Hannan, F. et al. (2012). " Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites." Hum Mol Genet 21(12):2768-2778. PubMed ID: 22422767
  • Hannan, F. et al. (2013). "Calcium-sensing receptor (CaSR) mutations and disorders of calcium, electrolyte and water metabolism." Best Pract Res Clin Endocrinol Metab 27(3):359-371. PubMed ID: 23856265
  • Human Gene Mutation Database (Bio-base).
  • Nesbit, M. et al. (2013). "Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3." Nat Genet 45(1):93-97. PubMed ID: 23222959
  • Nesbit, M. et al. (2013.) "Mutations affecting G-protein subunit α11 in hypercalcemia and hypocalcemia." N Engl J Med 368(26):2476-2486. PubMed ID: 23802516
  • Pearce, S. et al. (1995). "Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism." J Clin Invest 96(6):2683-2692. PubMed ID: 8675635
  • Pollak, M. et al. (1993). "Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism." Cell 75(7):1297-1303. PubMed ID: 7916660
  • Pollak, M. et al. (1994). "Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation." Nat Genet 8(3):303-307. PubMed ID: 7874174
  • Vargas-Poussou, R. et al. (2002). “Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome.” J Am Soc Nephrol 13(9):2259-2266. PubMed ID: 12191970
  • Vezzoli, G. et al. (2006). "Autosomal dominant hypocalcemia with mild type 5 Bartter syndrome." J Nephrol 19(4):525-528. PubMed ID: 17048213
  • Watanabe, S. et al. (2002). “Association between activating mutations of calcium-sensing receptor and Bartter's syndrome.” Lancet 360(9334):692-694. PubMed ID: 12241879
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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