Ruling in a Suspect: The Role of<i>AP2S1</i>Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Calcium homeostasis is regulated by PTH, and PTH secretion from parathyroid glands is inhibited by increases in extracellular calcium ([Ca2+]o) levels that are sensed by the calcium-sensing receptor (CaSR) (1). Primary hyperparathyroidism (PHPT) is a common endocrine disorder in which increased PTH secretion occurs despite increased [Ca2+]o, related in part to reduced CaSR expression. In familial hypocalciuric hypercalcemia (FHH), the hypercalcemia is accompanied by normal or elevated serum PTH levels, as in mild PHPT (2). However, renal and skeletal manifestations of PHPT are generally absent. A family history and calcium-to-creatinine clearance ratios that are low relative to the filtered load of calcium (<0.012) are important in distinguishing FHH from PHPT (1, 3). Roughly two-thirds of FHH cases are caused by heterozygous germline-inactivating mutations in the CASR gene (4, 5). In the parathyroid and renal tubular cells, where this G protein-coupled receptor (GPCR) is most abundantly expressed, direct calcium activation of the CaSR inhibits PTH secretion or decreases renal calcium reabsorption, respectively. Loss of function of one CASR allele in FHH reduces the sensitivity of the parathyroid and renal cells to calcium and leads to hypercalcemia and hypocalciuria (1). Homozygous loss-of-function mutations of the CaSR cause severe neonatal hyperparathyroidism that can be lethal without parathyroidectomy (6). Heterozygous germline-activating mutations in CASR underlie a subset of cases with autosomal dominant hypocalcemia/hypoparathyroidism (ADH) (1). These mutations cause increased parathyroid and renal sensitivity to calcium, such that PTH release is suppressed even at low calcium levels and there is relative hypercalciuria. In various kindreds, the FHH trait can be linked to chromosome 3q21.1, the CASR locus, and this is the more common FHH type 1 (4). However, genetic heterogeneity is also well-known. In one family, the trait mapped to chromosome 19p13.3 (FHH type 2), and in two other unrelated kindreds, the trait mapped to chromosome 19q13.3 (FHH type 3). Nesbit et al (7) have now shown that mutations in the AP2S1 gene underlie FHH3. They initially identified AP2S1 mutations by exome capture and high-throughput sequence analysis of genomic DNA from an affected individual in each of the two FHH3 kindreds. Although many previously unreported nonsynonymous single nucleotide variants were identified in the whole exomes of each subject, within the locus of the FHH3 trait, one single nucleotide variant—a C>T transition in exon 2 of the AP2S1 gene—was identified in affected individuals of both kindreds. The nucleotide substitution predicts a p.Arg15Cys missense alteration. Nesbit et al (7) next determined the frequency of AP2S1 mutations in 50 unrelated FHH cases in which CASR mutations had been excluded. Eleven missense heterozygous alterations, all affecting Arg15 (p.Arg15Cys, p.Arg15His, or p.Arg15Leu), were identified in patients whose ages ranged from 8 days to 78 years. The authors noted that all had similar elevations in serum calcium and PTH concentrations as well as reductions in urine calcium excretion. The AP2S1 gene encodes the σ-subunit of adaptor protein complex 2 (designated as the AP2σ2 protein; see Ref. 7) critical for clathrin-mediated endocytosis of a variety of cell-surface proteins including GPCRs. AP2 is a heterotetramer of α-, β-, μ-, and σ-subunits and links clathrin to vesicle membranes by binding to tyrosine and dileucine-based motifs of membrane-associated cargo proteins. An analysis of the crystal structure of AP2 shows that the Arg15 residue of AP2σ2 and the Arg21 residue of AP2α1 are involved in forming key contacts with a glutamine residue at position –4 relative to the first leucine of the dileucine motif of cargo proteins (7). Replacing the positively charged Arg15 residue with either the polar but uncharged Cys15 residue or the nonpolar Leu15 residue is predicted to lead to a reduced affinity for the dileucine motif and, hence, compromise receptor function. The effects of the His15 substitution are more difficult to predict. AP2 is known to have an important role in GPCR endocytosis and recycling, thereby potentially influencing GPCR sensitivity. CaSR has within its cytoplasmic C-terminal domain a variant dileucine motif (RHQPLL, residues 1009–1014). Nesbit et al (7) examined the effect of removing the CaSR dileucine motif by transiently transfecting HEK293 cells with either wild-type CaSR (Leu1013Leu1014) or mutant CaSR (Ala1013Ala1014) and measuring the intracellular calcium responses to changes in [Ca2+]o. Expression of the mutant CaSR led to a rightward shift in the concentration-response curve, with a higher EC50 value compared to wild-type, supporting the notion that the CaSR dileucine motif is functionally important. Expression of transiently expressed mutant (Cys15, Leu15, His15) AP2σ2 proteins in HEK293 cells stably expressing the CaSR led to a rightward shift in the concentration-response curves relative to those expressing the wild-type Arg15 AP2σ2 protein. This indicated a decrease in the sensitivity of cells expressing the FHH3-associated AP2S1 mutants relative to wild-type AP2S1. The effect of AP2S1 Arg15 alterations on CaSR cell-surface expression was evaluated under basal (0.5 mm [Ca2+]o) and stimulated (5.0 mm [Ca2+]o) conditions in HEK293 cells stably transfected with CaSR (7). Under short-term conditions (15 min) cell-surface expression of the CaSR was decreased in cells transfected with empty vector, and the AP2S1 wild-type decreased this level further, whereas the AP2S1 mutants were without any effect greater than that of empty vector. Hence, the AP2S1 mutants lacked the wild-type AP2S1 effect on CaSR endocytosis. Although the genetic evidence linking AP2S1 mutations to FHH3 is clear-cut, elucidation of the precise mechanism whereby AP2S1 Arg15 mutations cause impaired CaSR function will require further study. The work of Nesbit et al (7) supports the notion that endocytosis of the CaSR is important for activity. In apparent discord with this idea, however, CaSR mutations have been found in ADH cases that involve loss of a significant part of the C-terminal tail (eg, S895-V1075del), including the dileucine 1013,1014 motif (8). The higher cell-surface expression of the S895-V1075del mutant CaSR relative to wild-type had been postulated to be the basis for the greater activity and leftward shift in concentration-response assays relative to wild-type CaSR. In the present issue of the JCEM, Fujisawa et al (9) report on a female infant who was found to have idiopathic infantile hypercalcemia (IIH) after referral for poor weight gain on breast feeding. At 49 days of age, her corrected serum calcium, intact PTH, and urinary calcium-to- creatinine ratio were 13.1 mg/dL, 27 pg/mL, and 1.29, respectively. There was no evidence of hyperparathyroidism, PTHrP-producing neoplasm, or vitamin D excess. Except for hypercalciuria, the data were consistent with impaired CaSR signaling. With low calcium formula (2.6 mg/dL), the infant showed catch-up growth, and serum calcium and urinary calcium-to-creatinine ratio were decreased. However, serum PTH rose. Low-calcium and standard formula at a 2:1 ratio maintained serum calcium around 12 mg/dL without markedly increasing serum PTH. No pathological mutation was detected in the CASR gene, but a heterozygous p.Arg15Leu mutation was identified in the AP2S1 gene, the previously reported causative mutation for FHH3. Fujisawa et al (9) suggest that a lack of hypocalciuria does not necessarily argue against an AP2S1 mutation. Similarly, in a study of 32 undifferentiated IIH cases, we reported two infants with maternally inherited CASR-inactivating mutations but without hypocalciuria (10). As Fujisawa et al (9) suggest, the young age of the infant is likely playing a significant role in the variable occurrence, and possibly degree, of hypercalciuria. Important in this study is the evidence for efficacy of a low-calcium formula. The authors suggest that it may be of benefit in reducing the serum calcium level and, if used judiciously, helpful in restoring normal growth and development. Of further interest, Nesbit et al (11) have identified heterozygous germline mutations in GNA11, the gene encoding the α-subunit of G11, in affected members of a kindred with FHH2, and one unrelated FHH patient. GNA11 was selected using a candidate gene approach because it is located at 19p13.3, the locus of the FHH2 trait. In addition, Nesbit et al (11) and Mannstadt et al (12) identified four different heterozygous missense mutations in GNA11 in patients with ADH in whom CASR-activating mutations had not been detected. Gα11 mediates signaling by the CaSR; hence, GNA11-inactivating mutations would reduce and activating mutations would increase the sensitivity of the parathyroid gland and renal tubule to [Ca2+]o. Autosomal dominant hypocalcemia/hypoparathyroidism due to CASR mutations and GNAS11 are now designated as ADH type1 and type 2, respectively. Finally, it is now recognized that mutations of the 25-hydroxyvitamin D 24-hydroxylase gene (CYP24A1) also manifest in early infancy with a more severe symptomatic IIH phenotype that can include marked hypercalciuria and nephrocalcinosis (13–15). In conclusion, these recent studies give geneticists and clinical endocrinologists additional options in providing for molecular diagnostics in IIH and related PTH-independent disorders of calcium homeostasis (Table 1) in which CASR mutations have not been identified. Disorder, Chromosomal Locus, Gene Name, and Phenotype Abbreviations: HHC, hypocalciuric hypercalcemia, familial; HYPOC, hypocalcemia, autosomal dominant; MIM, Mendelian Inheritance in Man. Disorder, Chromosomal Locus, Gene Name, and Phenotype Abbreviations: HHC, hypocalciuric hypercalcemia, familial; HYPOC, hypocalcemia, autosomal dominant; MIM, Mendelian Inheritance in Man. This work was supported by the Canadian Institutes of Health Research (to G.N.H.) and the Dairy Farmers of Canada (to D.E.C.C.). Disclosure Summary: G.N.H. and D.E.C.C. have nothing to declare. autosomal dominant hypocalcemia/hypoparathyroidism adaptor protein complex 2 σ-subunit of AP2 gene encoding AP2σ2 extracellular calcium calcium-sensing receptor CaSR gene 25-hydroxyvitamin D 24-hydroxylase gene familial hypocalciuric hypercalcemia gene encoding α-subunit of G11 G protein-coupled receptor idiopathic infantile hypercalcemia primary hyperparathyroidism.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.