Hypercalcemic and Hypocalcemic Disorders via the CASR Gene

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.

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.

This test provides full coverage of all coding exons of the CASR gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.