4.4 Familial hypocalciuric hypercalcaemia

Familial hypocalciuric hypercalcaemia (FHH) is a generally asymptomatic form of mild to moderate, parathyroid hormone (PTH)-dependent hypercalcaemia, which was initially confused with the more common hypercalcaemic disorder, primary hyperparathyroidism (PHPT) (1–3). Subsequent studies showed that FHH differs from PHPT in several important respects, although distinguishing between these two conditions can still be difficult on a clinical basis alone (4). Urinary calcium excretion is lower in the former than in the latter, and in FHH, unlike PHPT, hypercalcaemia recurs rapidly following surgical treatment with anything less than total parathyroidectomy. Indeed, given FHH’s generally benign natural history, surgery is usually ill advised (3).

The phenotype of FHH implicated some abnormality in the sensing and/or handling of calcium by parathyroid and kidney (3, 5). For more than two decades after its initial description, however, the genetic defect in FHH was unknown. In 1992, the major genetic locus for this condition was identified on the long arm of chromosome 3 (6). The following year saw the cloning of a G protein-coupled extracellular calcium (Ca²⁺_o)-sensing receptor (CaSR) mediating direct regulation of PTH secretion by Ca²⁺_o (7). The CaSR’s function and its location of its gene on the same region chromosome 3 in humans made it an obvious candidate gene for FHH. Shortly thereafter, heterozygous inactivating mutations in the CaSR were identified in several FHH families (8). Moreover, patients with a related condition, neonatal severe hyperparathyroidism (NSHPT), also turned out to harbour inactivating CaSR mutations in the homozygous, compound heterozygous, and in a milder disorder, neonatal hyperparathyroidism (NHPT), in the heterozygous state (8). This chapter reviews the clinical and biochemical features of FHH, its genetics, pathophysiology, and pathogenesis, and its relationship to NSHPT.

In 1972, Foley, et al. described a family with an asymptomatic, unexpectedly benign form of hypercalcaemia that they called familial benign hypercalcaemia (9). Their report first detailed the distinctive clinical features of this syndrome, although in retrospect a family described in 1966 (10) proved to have the same condition when re-evaluated later. Subsequent work confirmed and refined these initial observations. Marx et al. studied families with the same syndrome and renamed it FHH to emphasize its characteristic alteration in renal Ca²⁺ handling (e.g. absolute or relative hypocalciuria, the latter being an inappropriately low urinary Ca²⁺ excretion in the face of hypercalcaemia) (3). The terms FHH and familial benign hypercalcaemia are both employed to describe this condition (or the hybrid term, familial benign hypocalciuric hypercalcaemia), but we shall use the first of these here.

FHH is an uncommon condition; its prevalence is thought to be 1% or less of that of PHPT. It exhibits an autosomal dominant inheritance of lifelong, generally asymptomatic hypercalcaemia of mild to moderate severity. FHH’s penetrance approaches 100% (3) and its biochemical abnormalities appear immediately postnatally. The degree of hypercalcaemia varies, but the serum calcium concentrations within a given family tend to be clustered within a relatively narrow range. Occasional families have serum calcium concentrations that are consistently within the upper part of the normal range or only intermittently elevated. Most families have serum total calcium concentrations of 2.6–2.9 mmol/l, while rare kindreds have values as high as 3–3.3 mmol/l or even higher (3, 11). Affected individuals do not, in general, exhibit the symptoms and complications of other hypercalcaemic disorders (1–3). These typical manifestations of hypercalcaemia include gastrointestinal (nausea, anorexia, and constipation), mental, and renal disturbances (nephrolithiasis, nephrocalcinosis, impaired renal function, and defective urinary concentrating capacity) (12). Even in FHH kindreds with unusually high serum calcium concentrations, affected individuals are remarkably asymptomatic. Nonspecific symptoms encountered in other hypercalcaemic disorders, such as fatigue, were initially reported in patients with FHH (3), but were not confirmed subsequently to be related to this condition and are thought to be the result of ascertainment bias (13).

Some persons with FHH have experienced pancreatitis or chondrocalcinosis, raising the possibility of a causal relationship between FHH and these complications (3). Some studies have found pancreatitis to be no more common in affected than in unaffected members of families with FHH or in the population as a whole (13). More recent studies, however, have suggested that the presence of FHH may increase the risk of pancreatitis in individuals with mutations in other genes, such as SPINK1, which by themselves confer increased risk of pancreatitis (14). In the case of chondrocalcinosis, follow-up studies failed to confirm that chondrocalcinosis occurs with increased frequency in FHH (13).

The degree of hypercalcaemia in FHH is comparable to that in mild to moderate PHPT, and both conditions exhibit equivalent increases in serum total and ionized calcium concentrations (3, 13). The serum phosphate concentration tends to be somewhat reduced in FHH but usually remains within the normal range. Serum magnesium is high-normal or mildly elevated. There is a positive relationship between the serum calcium and magnesium concentrations in FHH; PHPT, in contrast, exhibits an inverse relationship between these parameters (3).

A common abnormality in FHH is an inappropriately normal (i.e. nonsuppressed) PTH level or, less commonly, a mildly elevated level of this hormone (13), especially when measured with an intact PTH assay (6, 15). Thus Ca²⁺_o-regulated PTH release must be abnormal, since hypercalcaemia would otherwise suppress PTH secretion. One factor that can cause an unusually high level of PTH in FHH is coexistent vitamin D deficiency (16). It is in patients with FHH who have PTH levels that are in the upper part of the normal range or are frankly elevated that differentiating this condition from mild PHPT on the basis of serum calcium and PTH alone may be difficult (4), since 10–15% of hyperparathyroid patients exhibit intact PTH levels in the upper normal range and many have levels that are only mildly to moderately elevated.

Studies modulating serum calcium concentration in FHH by infusing calcium to raise it and citrate (or ethylenediamine tetraacetic acid (EDTA)) to lower it have revealed an increase in parathyroid ‘set-point’ (the level of Ca²⁺_o half-maximally suppressing PTH levels) (17). Thus FHH exhibits mild to moderate ‘resistance’ to the normal inhibitory effect of Ca²⁺_o on PTH release. PHPT exhibits an analogous, but somewhat greater, increase in set-point (17). PHPT also commonly exhibits additional defects in secretory control, including elevated maximal and minimal secretory rates at low and high Ca²⁺_o, respectively. The parathyroid glands in FHH appear normal or mildly hyperplastic (18), although occasional families have overt parathyroid enlargement and hyperplasia (19).

A number of individuals with FHH have undergone partial or total parathyroidectomy in an attempt to cure their hypercalcaemia, usually following an erroneous diagnosis of PHPT. Their unusual postoperative course has provided further evidence that FHH differs fundamentally from PHPT. Among 27 individuals with FHH who underwent from one to four neck explorations, hypercalcaemia recurred within days to weeks in most (21 patients), and only two remained normocalcaemic indefinitely without additional treatment (1). Cure of hypercalcaemia in FHH usually occurred only after total parathyroidectomy (5 of 27 persons). Recurrent hypercalcaemia after resecting a parathyroid adenoma, in contrast, occurs in less than 5–10% of cases, usually several years postoperatively. Recurrent hypercalcaemia is more common in primary parathyroid hyperplasia, particularly in familial disorders, such as multiple endocrine neoplasia type 1 (MEN 1). The incidence of recurrence in the latter condition increases progressively to approximately 50% at 10 years after subtotal parathyroidectomy (20).

Serum 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH)₂D) levels are generally normal in FHH (15), and intestinal Ca²⁺ absorption is normal or modestly reduced (13). Some persons with FHH show a blunted rise in 1,25(OH)₂D and gastrointestinal Ca²⁺ absorption when dietary calcium intake is reduced (13). Patients with PHPT exhibit higher levels of 1,25(OH)₂D than those in patients with FHH (15), accompanied by increased calcium absorption. Markers of bone turnover (i.e. urinary deoxypyridinoline excretion) can be mildly elevated in FHH, but bone mineral density is generally normal (21) and is higher in the hip and forearm—areas relatively rich in cortical bone—than in patients with PHPT, who typically exhibit loss of cortical bone. As might be expected from their bone mineral density, fracture risk is not increased in FHH patients (13). Several affected persons in an FHH kindred in Oklahoma, USA exhibited osteomalacia (22). However, osteomalacia is not a feature of other FHH kindreds, and this Oklahoma kindred has a form of FHH that is genetically distinct from that in most kindreds (see below).

Another characteristic finding in FHH is excessively avid renal tubular reabsorption of Ca²⁺ and Mg²⁺ (Fig. 4.4.1a) (3, 9, 13), particularly given the concomitant hypercalcaemia, which normally increases urinary Ca²⁺ excretion (12). The parameter of renal Ca²⁺ handling utilized most frequently to document this abnormality is the ratio of the clearance of calcium to that of creatinine, calculated as (urinary calcium/ serum total calcium)×(serum creatinine/ urinary creatinine). This clearance ratio is lower than 0.01 in approximately 80% of individuals with FHH but in only about 20% of patients with PHPT (23). Persons with other, non-PTH-dependent forms of hypercalcaemia generally exhibit markedly greater rates of calcium excretion. In a recent study, a calcium to creatinine clearance ratio of 0.0115 provided 80% sensitivity and 88% specificity in distinguishing FHH from PHPT (24). Thus the clinical constellation of autosomal dominant inheritance of mild, asymptomatic hypercalcaemia in two or more first-degree family members, a low urinary calcium to creatinine clearance ratio, and a normal PTH level usually makes the diagnosis of FHH straightforward. The excessive renal tubular reabsorption of Ca²⁺ in FHH patients exhibiting the usual hypocalciuric phenotype persists even after total parathyroidectomy (5), showing that it is not dependent upon PTH but is an intrinsic defect in renal sensing/ handling of Ca²⁺ (Fig. 4.4.1b).

Confusion can arise in distinguishing patients with FHH from those with mild PHPT in the setting of conditions that would be expected to lower urinary calcium excretion in PHPT, such as vitamin D deficiency, very low calcium intake, concomitant use of thiazide diuretics, coexistent hypothyroidism, or during treatment with lithium for psychiatric disorders (lithium can also predispose to and/or unmask PTH-dependent hypercalcaemia) (2, 12). Moreover, in persons with PHPT and greater than a 50% reduction in glomerular filtration rate owing to chronic renal dysfunction, urinary calcium excretion decreases due to the renal insufficiency per se. Use of the calcium to creatinine clearance ratio to distinguish between FHH and PHPT may be of limited utility in the circumstances just noted, although correction of coexistent medical conditions, e.g. vitamin D or calcium deficiency, and studies of additional family members may clarify the diagnosis. Moreover, as discussed later, genetic testing is appropriate in some settings for unequivocal diagnosis of FHH. An additional parameters of renal function that is altered in FHH is urinary concentrating ability. While hypercalcaemia of other causes can produce defective urinary concentrating ability (25), individuals with FHH concentrate their urine to a greater extent than do patients with PHPT who have a comparable degree of hypercalcaemia (26).

In occasional FHH kindreds, hypercalcaemia has accompanied by hypercalciuria and even overt renal stone disease (27, 28). In one such kindred, in which FHH was caused by the most common genetic form of FHH linked to chromosome 3 (see Genotype–phenotype relationships, below), hypercalciuria and/or nephrolithiasis were present in several affected family members and were corrected in most cases by subtotal parathyroidectomy (27). The parathyroid glands in this family differed from the norm in FHH in that many revealed nodular hyperplasia.

Thus both the clinical and biochemical manifestations of FHH suggested that it was an inherited abnormality in the responsiveness of parathyroid, kidney, and perhaps other tissues to Ca²⁺_o. In the latter regard, there is a notable lack, for instance, of the usual gastrointestinal or mental symptoms of hypercalcaemia in FHH, even in kindreds with higher than average serum calcium concentrations (3, 9, 13). Given the benign natural history of FHH and the difficulty in achieving a biochemical ‘cure’, a consensus has emerged that surgical intervention is unwise in this condition except in unusual circumstances detailed below. Therefore, differentiating FHH from PHPT is very important to avoid unnecessary neck exploration in the former.

Studies converging from two different directions established, on the one hand, that the extracellular CaSR is a key player in the maintenance of extracellular Ca²⁺ homoeostasis while, on the other hand, also representing the disease gene for the most common form of FHH. By briefly describing the biochemistry and biology of the CaSR and how it maintains Ca²⁺_o homoeostasis, this section provides a foundation for the ones that follow detailing the molecular genetics and pathophysiology of FHH.

Expression cloning in Xenopus laevis oocytes enabled isolation of the CaSR from bovine parathyroid (7). The bovine CaSR and the same receptor in other mammalian species, including humans, have three key structural domains: The first is a large N-terminal extracellular domain (ECD) comprising over 600 amino acids. The second comprises an approximately 250 amino acid transmembrane domain (TMD) that includes seven transmembrane helices, and three extracellular and three intracellular loops. These structural features of the TMD are characteristic of the large superfamily of G protein-coupled receptors (GPCR). The last domain is the CaSR’s approximately 200 amino acid cytoplasmic, carboxyl (C)-terminal tail. The CaSR resides on the cell surface as a disulfide-linked dimer (29). Sensing of Ca²⁺_o occurs largely within its ECD (30), although elements within the TMD probably also participate in Ca²⁺_o-sensing, as a ‘headless’ CaSR, totally lacking its ECD, retains some responsiveness to Ca²⁺_o. Changes in the conformations of the ECD, transmembrane helices and extra- and/or intracellular loops occurring following binding of extracellular Ca²⁺_o to the CaSR are thought to activate G proteins (especially G_q/11 and G_i) and enable the receptor to couple its intracellular effector systems. These comprise numerous signalling cascades, including activation of phospholipases C, A₂, and D and mitogen-activated kinases (MAPK) and inhibition of adenylate cyclase (31). The relative contributions of these signalling pathways to the CaSR’s biological actions in its various target tissues remain to be fully elucidated in most cases.

CaSR-expressing tissues with clear homoeostatic roles include the parathyroid chief cells, thyroidal C-cells, and kidney (32). The CaSR is also expressed in bone cells, including osteoblasts, osteoclasts, and osteocytes, but its physiological roles in these cell types remain somewhat controversial and are the subject of active investigation. Available data, however, support physiologically relevant roles of the CaSR in promoting osteoblastic bone formation and in inhibiting osteoclastogenesis and osteoclastic bone resorption, physiological functions that have been recently reviewed in detail elsewhere (33).

In the parathyroid, activating the CaSR inhibits PTH secretion, parathyroid cellular proliferation, and PTH gene expression (34). In C-cells, in contrast, the CaSR stimulates, rather than inhibiting, hormonal secretion (e.g. of calcitonin) (34). Since PTH is a Ca²⁺_o-elevating hormone and calcitonin a Ca²⁺_o-lowering hormone, the CaSR-mediated inhibition of PTH secretion and stimulation of calcitonin secretion are homoeostatically essential for defending against hypercalcaemia. Conversely, stimulation of PTH secretion is a key defence against hypocalcaemia.

The CaSR is present along most of the renal tubule, including proximal convoluted and straight tubules, medullary and cortical thick ascending limbs (MTAL and CTAL, respectively), distal convoluted tubule, and cortical, outer medullary, and inner medullary collecting ducts (35). In CTAL, which synthesizes the highest level of the CaSR in the kidney, the receptor resides principally on the basolateral cell surface, where it senses systemic (i.e. blood) levels of Ca²⁺_o. The CaSR in CTAL, and perhaps also in the distal convoluted tubule, directly regulates tubular Ca²⁺ and Mg²⁺ handling, increasing their reabsorption when Ca²⁺_o is low and diminishing it if Ca²⁺_o is high (36). The CaSR and the PTH receptor are both expressed in CTAL, where they antagonize one another’s actions on Ca²⁺ reabsorption—the CaSR inhibiting and the PTH receptor enhancing it. In the inner medullary collecting ducts, the CaSR is on the apical (e.g. luminal) plasma membrane and monitors Ca²⁺_o within the urine (37). This apical CaSR probably mediates the high Ca²⁺_o-evoked decrease in vasopressin-stimulated water reabsorption noted above (36). This action could potentially reduce the risk of forming renal stones when urinary Ca²⁺ is high. The CaSR probably also diminish maximal urinary concentration by inhibiting NaCl reabsorption in the MTAL, thereby reducing the medullary hypertonicity needed to drive vasopressin-stimulated water reabsorption in the collecting duct (36).

To summarize, the CaSR’s roles in defending against hypercalcaemia include the following: High Ca²⁺_o inhibits PTH secretion, which reduces net release of Ca²⁺ from bone owing to the fact that PTH is a stimulator of bone resorption. Decreased PTH release also has two key effects on the kidney, enhancing renal Ca²⁺ excretion and reducing proximal tubular synthesis of 1,25(OH)₂D₃, both of which are normally enhanced by PTH. The reduced synthesis of 1,25(OH)₂D decreases gastrointestinal absorption of Ca²⁺. As a result, there is decreased influx of Ca²⁺ into the extracellular fluid from intestine and bone and increased excretion of Ca²⁺ via the kidneys, thereby normalizing Ca²⁺_o. Additional consequences of high Ca²⁺_o-elicited activation of the CaSR that contribute to the defence against hypercalcaemia include stimulation of calcitonin, direct inhibition of distal renal tubular Ca²⁺ reabsorption in the CTAL, direct inhibition of 1-hydroxylation of 25(OH)D in the proximal tubule, and, perhaps, CaSR-mediated stimulation of osteoblastic activity and inhibition of osteoclastic function (33). Hypocalcaemia elicits reciprocal changes in these various parameters, permitting an effective defence against hypocalcaemic challenges.

While the preceding is a well-accepted description of the body’s homoeostatic responses to hyper- and hypocalcaemia, recent studies utilizing mice with knockout of the CaSR suggest that low Ca²⁺_o-evoked, CaSR-mediated enhancement of PTH secretion may serve primarily to defend against hypocalcaemia, in effect acting as a homoeostatic ‘floor’, and play a less essential role in defending against hypercalcaemia. CaSR-mediated inhibition of renal Ca²⁺ reabsorption and stimulation of calcitonin secretion (although the latter may be less important in humans than in calcitonin-responsive species such as rodents), in contrast, may be key elements of the homoeostatic ‘ceiling’ defending against hypercalcaemia (Kantham, et al., in press).

Chou et al. first mapped the FHH disease gene in four families to the long arm of chromosome 3 (q21–24) (6), although this locus has subsequently been refined to 3q13.3-q21 (see http://www.casrdb.mcgill.ca). Identification of this genetic locus made it possible to show, using closely linked genetic markers, that persons with FHH are heterozygous for the disease gene (38). Subsequent studies demonstrated that most (c. 90% or more) FHH kindreds sufficiently large for genetic analysis exhibit linkage to chromosome 3. This genetic form of FHH is called hypocalciuric hypercalcaemia, type 1 (HHC 1, OMIM 145980) in the Online Mendelian Inheritance in Man (OMIM) database. In one family, however, a disorder clinically indistinguishable from FHH was linked to the short arm of chromosome 19, band 19p13.3 (39), and this variant of FHH is termed HHC 2 (OMIM 145981). Moreover, the Oklahoma kindred mentioned earlier with unusual clinical features (e.g. osteomalacia and rising PTH levels with age) was shown to be linked to chromosome 19, band q13 (22), and this variant of FHH is called HHC 3 (OMIM 600740). Therefore, FHH is genetically heterogeneous, but only the disease gene causing HHC 1 has been identified (see next section). A severe neonatal form of hyperparathyroidism (neonatal severe hyperparathyroidism (NSHPT)) is sometimes encountered in FHH kindreds (38). It represents the homozygous form of FHH linked to chromosome 3 in most cases. The clinical, biochemical, and genetic features of NSHPT are described below.

Because of the abnormal Ca²⁺_o-sensing by kidney and parathyroid in FHH, the CaSR was a good candidate for the disease gene. Pollak et al. showed that point mutations (i.e. a change in a single nucleotide base producing a nonconservative change in the receptor’s coding sequence) were present in the CaSR gene in three FHH families that were linked to chromosome 3 (Fig. 4.4.2) (8). Subsequent studies have identified nearly 200 CaSR mutations in kindreds with FHH, many of which can be accessed at http://www.casrdb.mcgill.ca/. Generally, each family harbours its own unique mutation, although several mutations have recurred in apparently unrelated kindreds (e.g. p.R185Q, p.P55L, p.T138M, and p.T151M—in current terminology p.R185Q designates mutation of the arginine at amino acid 185 in the CaSR protein sequence to glutamine) (http://www.casrdb.mcgill.ca/). Most mutations are missense mutations (a new amino acid is substituted for the one normally coded for) (21, 30–33, 35), but additional types of mutations that have been identified include: (1) nonsense mutations (e.g. point mutations introducing a stop codon), (2) frame shift mutations (loss or gain of one or more nucleotides, thereby modifying the downstream coding sequence), (3) insertion of a substantial segment of unrelated nucleotide sequence (e.g. an Alu repetitive element), and (4) a mutation of a splice site at the CaSR gene’s intron–exon boundaries (for summary, see http://www.casrdb.mcgill.ca/). These mutations reside throughout most of the receptor’s amino acid sequence.

Mutations within the CaSR coding region have only been identified, however, in about two-thirds of FHH families linked to the locus on chromosome 3 (although linkage analysis has been carried out in only a minority of FHH families). The remaining families presumably have mutations in other areas of the gene, such as regulatory regions, which impact its level of expression, but further studies are needed. Several polymorphisms reside within the CaSR coding region or within the intervening sequences between coding exons (http://www.casrdb.mcgill.ca). Some studies have shown subtle effects of polymorphisms within the CaSR C-tail on parameters such as serum calcium concentration (40), urinary calcium excretion (41), or the severity of hyperparathyroidism, but these observations have not always been reproducible.

The ability to identify FHH by genotype, rather than just by phenotype, has considerably expanded the spectrum of clinical presentations resulting from inactivating mutations of the CaSR. About 15–20% of kindreds thought to have familial isolated PHPT have been shown to harbour inactivating mutations of the CaSR gene (28). Members of these kindreds present with a clinical picture typical of PHPT, without the characteristic relative or absolute hypocalciuria of FHH. It remains to be determined whether specific functional properties of the CaSR mutations in these kindreds can explain their clinical presentation and whether their clinical management should differ from that of typical FHH. The family described earlier with hypercalciuria and kidney stones (27) was shown to have a mutation within the CaSR C-tail and represents an example of an FHH kindred with features of familial isolated hyperparathyroidism.

Several cases of FHH have presented with coexistent parathyroid adenomas and more marked hypercalcaemia than is the norm in FHH (42). Removal of the adenoma produced a return of serum calcium concentration to a level more characteristic of FHH. It is not currently known whether the presence of FHH caused a predisposition to the development of an adenoma in these cases or the latter was coincidental. Some infants with heterozygous inactivating mutations of the CaSR present with hyperparathyroid bone disease, high PTH levels and moderate hypercalcaemia that is more severe than in typical FHH but less severe than is usually encountered in NSHPT (43). Such cases have been termed neonatal primary hyperparathyroidism (NHPT) and not infrequently revert to a picture compatible with FHH following conservative medical management or occasionally after partial parathyroidectomy (32). The clinical features of NSHPT and NHPT are discussed in more detail below. Finally, cases have been described of individuals homozygous for inactivating mutations of the CaSR that were only identified serendipitously in adulthood (44). Despite serum calcium concentrations, presumably lifelong, in the range of 3.75 mmol/l, these individuals were remarkably asymptomatic. They appear to harbour CaSR mutations sufficiently mild in their functional impairment to permit the affected individuals to survive undetected throughout childhood. This broader spectrum of clinical manifestations of inactivating CaSR mutations makes it important for the clinician to remain vigilant in order to correctly diagnose such patients.

Transient transfection in human embryonic kidney (HEK293) cells has been utilized to express CaSRs harbouring a number of the mutations identified in FHH kindreds (11). These studies have suggested several mechanisms through which these mutations alter not only the function of the mutated receptor but also that of the normal CaSR, which coexists in the cells of persons with FHH since it is a heterozygous condition.

Figure 4.4.3 illustrates the impact of several missense mutations on high Ca²⁺_o-evoked increases in the cytosolic calcium concentration. Mutations within the CaSR ligand-binding ECD probably interfere with its activity via two mechanisms: (1) by reducing the mutant receptors’ affinity for calcium (10) and/or (2) by decreasing its cell surface expression. The mutation, p.R185W, for example, markedly reduces both maximal response and apparent affinity of the CaSR (11) without substantially lowering its cell surface expression. However, as discussed in more detail later, this mutation’s negative impact extends beyond its effect on the mutant receptor, because it also exerts a dominant negative action on the coexpressed normal receptor (11).

Some mutant CaSRs exhibit almost complete loss of biological activity owing to a markedly reduced level of cell surface expression. For instance, the mutation, p.R66C, creates an unpaired cysteine within the ECD that probably forms mispaired intra- and/or intermolecular disulfide bonds, thereby producing a structurally distorted receptor protein(s) that fails to reach the cell surface (11). Mutant CaSRs may also: (1) fail to enter the endoplasmic reticulum from their ribosomal site of synthesis because of missense mutations within the CaSR signal peptide—the latter is needed for translocation of the nascent receptor protein into the endoplasmic reticulum (45); (2) fail to exit the endoplasmic reticulum and are degraded (46); or (3) leave the endoplasmic reticulum but encounter a biosynthetic block at the level of the Golgi apparatus (46). Another class of mutations severely reducing biological activity is nonsense mutation, because the resultant truncated receptor protein lacks structural determinants needed for biological activity.

Mutations within the CaSR transmembrane domains, extracellular loops, intracellular loops, or C-terminal tail probably also impact negatively on the receptors’ function through several mechanisms: (1) Mutations producing truncated CaSRs, such as a frame shift mutation at codon 747, produce receptors with gross structural alterations that both abolish biological activity and severely diminish cell surface expression; and (2) missense mutations may interfere with steps involved in receptor signalling, e.g. the mutation, p.R795W, within the CaSR third intracellular loop, probably interferes directly with G protein binding and/or activation (11).

Of interest, the degree of elevation in serum calcium concentration in families with CaSR mutations greatly reducing its cell surface expression (i.e. p.R66C) and/or producing nonfunctional receptors (e.g. p.S607X—where X refers to a stop codon) (43) can be relatively mild (e.g. ≤0.25 mmol/l) above that of unaffected family members. Conversely, mutant receptors exhibiting robust cell surface expression, such as p.R185Q and p.R795W, can cause more severe hypercalcaemia (11). How can these observations be explained? Several developments have provided significant insights into the factors contributing to the severity of hypercalcaemia in FHH. First, the cell surface form of the receptor is known to be a disulfide-linked dimer (29) and, second, the development of mice with targeted disruption (e.g. ‘knockout’) of one allele of the CaSR gene has provided a useful animal model of FHH (47).

In contrast to null mutations, the p.R795W or p.R185Q mutations produce serum calcium concentrations in affected family members that are 0.5 mmol/l and 0.75 mmol/l higher, respectively, than in unaffected family members (11). These mutations exert a so-called dominant negative effect on the wild type receptor when the two CaSRs are cotransfected. That is, coexpression of receptors bearing the p.R795W or p.R185Q mutation with the wild type CaSR (to mimic the heterozygous state in FHH) causes a rightward shift in the EC₅₀ relative to that of the normal receptor (the concentration of agonist evoking half of the maximal response) (Fig. 4.4.4) (11). In contrast, cotransfection of the normal CaSR with mutant receptors whose cell surface expression is greatly reduced often has much less or no effect on the normal receptor’s function. This dominant negative action results from the formation of heterodimers of the wild type and mutant receptors. Since the CaSR normally functions as a dimer, if these heterodimers are less active than wild type homodimers, then the number of normally functioning CaSRs on the cell surface (e.g. homodimers of the normal receptor) will be less than when the normal CaSR is cotransfected with mutant CaSRs functioning as null mutants. In other words, on a purely statistical basis, the proportion of wild type homodimers, heterodimers, and mutant homodimers in the former situation will be 1:2:1 (i.e. 25% wild type homodimers, 50% wild type-mutant heterodimers, and 25% mutant homodimers), and the 25% wild type homodimers will be the only normally functioning form of the CaSR present on the cell surface. In contrast, with true null mutations, the mutant receptor is not present and will not interfere with the function of the wild type receptor homodimers arising from the remaining normal CaSR-encoding allele.

In heterozygous CaSR knockout mice, the levels of CaSR expression in parathyroid and kidney are about 50% of those in wild type (i.e. normal) mice (47). Thus loss of one CaSR allele does not produce any substantial increase in the expression of the remaining normal allele, and the 50% reduction in CaSR expression is associated with mild PTH-dependent hypercalcaemia and relative hypocalciuria. The pathophysiology of the heterozygous mice appears to be similar to that in FHH families with ‘null’ mutations. Presumably in these patients, as in the heterozygous mice, the reduced complement of normal CaSRs resulting from loss of one CaSR allele causes a mild increase in parathyroid set-point and increased renal tubular reabsorption of calcium with resultant mild hypercalcaemia.

In the family described earlier with a mutation in the CaSR C-terminal tail, it is possible that this particular mutation more substantially reduces the mutant receptor’s function and/or cell surface expression in parathyroid than in kidney (27). That is, the kidney might respond more normally to Ca²⁺_o than the parathyroid in affected family members, producing PTH-dependent hypercalcaemia in association with hyper- rather than hypocalciuria. Thus, while we remain at a relatively early stage in our ability to predict phenotype from genotype, specific examples now exist of individual mutations that modify the degree of elevation in the serum calcium concentration and/or urinary calcium excretion. It should be pointed out, however, that among the nearly 200 FHH mutations that have been described to date, there is a large degree of overlap in serum and urine parameters, and, in the majority of these mutations, studies of the respective receptors’ functional properties in vitro, including the use of cotransfection with the mild type receptor, have not yet been performed.

With the identification of the CaSR gene as the disease gene in the most common form of FHH came the ability to perform genetic testing to confirm the diagnosis in probands and other affected family members. When should genetic testing be carried out? As noted above, the constellation of asymptomatic, mild hypercalcaemia with an autosomal dominant pattern of inheritance, a normal serum PTH level, and a urinary calcium to creatinine clearance ratio of 0.01 or less is essentially diagnostic of FHH. No further evaluation is warranted in such cases. There are several instances, however, in which genetic testing is appropriate. These include: (1) apparently sporadic or de novo cases of FHH or those who do not have any other family members available for testing, (2) affected members of kindreds with familial isolated hyperparathyroidism, as approximately 15–20% of such kindreds have mutations in the CaSR, and (3) as described in more detail in the next section, in children shown to have PTH-dependent hypercalcaemia prior to the age of 10 years.

Experience with a large number of FHH kindreds indicates that conservative medical follow-up, similar to that used to follow patients with asymptomatic PHPT, is an appropriate course of action (1, 3, 13). The rare instances in which parathyroid surgery might be contemplated are: (1) in cases in which hypercalcaemia and the degree of elevation in PTH are unusually severe, particularly if accompanied by hypercalcaemic symptoms and/or complications of hyperparathyroidism (e.g. bone disease, kidney stones), including patients with CaSR mutations presenting as FIH; and (2) perhaps in patients with recurrent pancreatitis who have mutations in other genes predisposing to pancreatitis (i.e. the SPINK1 gene). The calcimimetic, cincalcet, is an allosteric activator of the CaSR, which is approved as a medical therapy for severe hyperparathyroidism in patients receiving dialysis treatment for kidney failure (49) or for parathyroid cancer (in the USA) as well as for PHPT in Europe. It provides a novel medical therapy of potential utility in patients with FHH being considered for parathyroid surgery.

NSHPT presents at birth or shortly thereafter, often during the first week of life (1, 32, 50), with varying combinations of anorexia, constipation, failure to thrive, hypotonia, and respiratory distress. Respiratory compromise can be due to thoracic deformity, sometimes owing to a flail chest syndrome resulting from multiple fractures of severely demineralized ribs (1, 50). Hypercalcaemia in NSHPT can be severe, on the order of 3.5 to 5 mmol/l, and levels as high as 8 mmol/l have been recorded. Serum magnesium concentrations, when available, have sometimes been well above the normal range (1, 32). Serum PTH is often 5–10-fold elevated, although the increase can be more modest (1, 32, 50). Despite marked hypercalcaemia, affected infants can exhibit relative hypocalciuria. Skeletal radiographs frequently show profound demineralization, fractures of long bones and ribs, metaphyseal widening, subperiosteal erosion, and occasionally rickets (50). Skeletal histology reveals typical osteitis fibrosa cystica of severe hyperparathyroidism. All four parathyroid glands are enlarged and exhibit chief cell or water-clear cell hyperplasia (1, 50).

Before 1982 (1), NSHPT often had a fatal outcome without a prompt and aggressive combination of medical and surgical treatment. More recent series have described infants with neonatal hyperparathyroidism and hyperparathyroid bone disease but less severe hypercalcaemia (2.75–3 mmol/l) (43). Moreover, these cases can run a self-limited course with medical therapy alone, exhibiting healing of bone disease and reversion to a milder form of hypercalcaemia resembling FHH after several months (43). The genetic basis for this less severe form of neonatal hyperparathyroidism (NHPT) is described below. In symptomatic cases of NSHPT, initial management should include vigorous hydration, inhibitors of bone resorption such as the bisphosphonate, pamidronate, and respiratory support. It should be emphasized that each patient must be treated individually, as even several patients homozygous for FHH mutations within the same family may have varying degrees of clinical severity. If the infant’s condition is very severe or deteriorates during medical therapy, total parathyroidectomy within the first month of life with autotransplantation of a portion of one gland is usually recommended (1). Some authors recommend total parathyroidectomy followed by management of the resultant hypoparathyroidism using calcium and vitamin D supplementation to prevent symptomatic hypocalcaemia (50). The activity of many mutant CaSRs is enhanced by calcimimetics. Accordingly, a potential addition to the other modalities of the therapy for NSHPT is the use of cinacalcet to determine whether it is capable of lowering the serum calcium concentration.

Infants with NSHPT were described in FHH kindreds, suggesting that the former could be the homozygous form of the latter (50). Pollak et al. (38) utilized genetic markers to show that NSHPT was the homozygous form of FHH in three families with consanguineous marriages. Since homozygous infants have no normal CaSR genes, they manifest much more severe clinical and biochemical manifestations as a result of marked ‘resistance’ to Ca²⁺_o than in FHH. NSHPT can also be caused by compound heterozygous CaSR mutations, i.e. the inheritance two different CaSR mutations from two unrelated parents (51). Not surprisingly, having no normal CaSRs, this infant, similar to those with homozygous FHH, exhibited severe hypercalcaemia (6.6 mmol/l). Mutational analysis of the CaSR gene in NSHPT is important to document the presence of CaSR mutations. The parents should also be tested and receive appropriate genetic counselling regarding the risk that future offspring will be affected with FHH or NSHPT.

As noted above, a clinical picture has been described in the neonatal period and during early childhood that is intermediate in severity between the usual asymptomatic presentation of FHH and the marked hypercalcaemia, hyperparathyroidism and bone disease of NSHPT. Such infants have proven in some cases to be heterozygous for inactivating CaSR mutations. Why do infants with NHPT present with a more severe phenotype than those with typical FHH? A factor potentially contributing to NHPT in a heterozygous child with an affected father and an unaffected mother is the impact of normal maternal calcium homoeostasis on the fetus’ abnormal Ca²⁺_o-sensing in utero. Calcium is transported actively across the placenta from mother to fetus, producing a higher fetal than maternal calcium concentration. Therefore, a normal mother would expose her affected fetus’ parathyroid glands to a level of Ca²⁺_o that would be sensed as ‘hypocalcaemic’ by the latter. ‘Overstimulation’ of the fetal parathyroids would then ensue, causing superimposition of secondary fetal/ neonatal hyperparathyroidism on top of the abnormal Ca²⁺_o-sensing already present. Support for this hypothesis has been provided by the occurrence NSHPT in cases where the father had FHH and the mother appeared normal (1). Postnatally, the ‘secondary’ hyperparathyroidism would gradually resolve over several months, eventually reverting to typical features of FHH. Most children with FHH born to normal mothers, however, do not manifest more severe hypercalcaemia than those born to affected mothers. Some FHH families may be more susceptible to the development of NHPT in heterozygous infants because their mutant CaSRs exert a dominant negative effect (48). A third contributory factor might be the presence of vitamin D deficiency in the mother and/or her infant.

The discovery of the CaSR proved that extracellular calcium ions can act in a hormone-like fashion via their own cell surface, G protein-coupled receptor. In other words, the cells and tissues expressing the CaSR can communicate with one another using Ca²⁺_o as an extracellular first messenger. A corollary is that FHH and NSHPT are disorders of reduced hormone action, analogous to better recognized hormone resistance syndromes, such as androgen or insulin resistance (52). FHH is a condition in which the target tissues expressing the CaSR are mildly to moderately ‘resistant’ to the actions of Ca²⁺_o, while the Ca²⁺_o-resistance in NSHPT is moderate to severe. Both are examples of generalized Ca²⁺_o-resistance, while PHPT exhibits ‘tissue-selective’ Ca²⁺_o-resistance (i.e. only the pathological parathyroid gland(s) show reduced responsiveness to the Ca²⁺_o). Furthermore, although not discussed here (see Chapter 4.6), activating mutations in the CaSR have been identified as a cause of sporadic and, in some cases, an autosomal dominant form of hypocalcaemia associated with relative hypercalciuria (53). In contrast to FHH and NSHPT, these hypocalcaemic syndromes represent generalized ‘over responsiveness’ or ‘oversensitivity’ to Ca²⁺_o. They are analogous to the rapidly expanding group of disorders caused by activating mutations in various other types of receptors (54).