Distribution and Metabolic Actions of Calcium

The total body content of calcium in a normal adult is approximately 1000 g, of which nearly all exists within the crystal structure of bone mineral; less than 1% is soluble in extracellular and intracellular fluid.^1,2 On average, bone mineral closely approximates the composition of hydroxyapatite [Ca₁₀(PO4)₆(OH)₂], which means that 6 mmol of phosphate are released with every 10 mmol of calcium mobilized during bone resorption (or about 1 : 2 on a mg/mg basis).^1,3

In blood, calcium is partly bound to proteins. Albumin accounts for about 70% of the protein-bound fraction.⁴ Another portion (about 6%) is associated with diffusible ion complexes.¹ Thus normally, only half of total plasma calcium is freely ionized, but it is this fraction that is most important physiologically and subject to stringent endocrine regulation. The normal ranges of total and ionized serum calcium in the adult are 8.5 to 10.5 mg/dL and 1.17 to 1.33 mmol, respectively.⁵ The usual 2 : 1 ratio of total to ionized calcium may be disturbed by disorders such as metabolic acidosis (calcium binding to proteins is reduced at acid pH) or by changes in serum protein concentrations, as in starvation, cirrhosis, dehydration, or multiple myeloma. When precise knowledge of the ionized calcium concentration is clinically important, direct measurement with calcium-selective electrodes should be performed.

Serum calcium is higher during infancy/childhood and adolescence than in the adult⁶ but does not change at puberty or, in women, during the menstrual cycle. During pregnancy, total serum calcium and albumin decline progressively, but ionized calcium is minimally affected.^7,8 Fetal calcium content rises dramatically during the third trimester (to about 30 g at term),⁹ and fetal serum total and ionized calcium concentrations both are higher than maternal levels, consistent with active placental transport of calcium.¹⁰ Despite daily losses of 200 to 300 mg/d of calcium in breast milk, lactating women maintain normal levels of ionized calcium in blood by increasing intestinal calcium absorption in response to augmented production of 1,25(OH)₂D.^11,12

Calcium entry into cells is strongly favored by a steep electrochemical gradient. Thus, the cell interior is electronegative, and cytosolic free calcium is in the range of 10⁻⁷ M, which is 10,000-fold lower than extracellular calcium concentrations. Calcium traverses the plasma membrane through various channels, including voltage-, receptor- and store-operated forms, the regulation of which is complex and tissue specific.^13,14 Intracellularly, nearly all (99%) calcium is sequestered in pools within mitochondria, endoplasmic reticula, or sarcoplasmic reticula or is tightly bound to the inner surface of the plasma membrane.¹⁵ Sequestered calcium, especially that within the endoplasmic reticulum, may be released rapidly into the cytosol following activation of cell-surface receptors. In this way, it plays a critical role in signal transduction and in controlling calcium entry via store-operated channels. The extremely low cytosolic free calcium concentration is maintained by active calcium transport into intracellular pools or by extrusion out of the cell via high-affinity, low-capacity Ca²⁺/H⁺ adenosine triphosphatases (ATPases) and low-affinity, high-capacity Na⁺/Ca²⁺ exchangers driven by the transmembrane sodium gradient.¹⁶

With the discovery of a G protein–coupled calcium-sensing receptor (CaSR) expressed in parathyroid, renal epithelial, and other cells, it has become clear that calcium can act as an extracellular ligand to directly control cellular function.¹⁷ The principal actions of the CaSR with respect to calcium homeostasis include suppression of PTH secretion and, in the thick ascending loop of the renal tubule, inhibition of calcium, magnesium, and NaCl reabsorption. Extracellular calcium also is utilized directly for normal matrix mineralization in bone and cartilage and is required for activation of important circulating or extracellular enzymes and proteases. Intracellular calcium exerts a broad range of effects via interaction with key enzymes and effector molecules, including kinases, phosphatases, calmodulins, transcription factors, ion channels (including calcium channels), and troponins and other proteins involved in contraction, microtubule and microfilament assembly, and motility. The steep gradients between intracellular calcium and both extracellular and intracellularly sequestered calcium are crucial for normal neuromuscular activity and provide the potential required for rapid transients and waves of cytosolic free calcium that serve key second-messenger functions in both excitable and nonexcitable cells.^14,18

Calcium Absorption

Intestinal calcium absorptive efficiency in humans ranges broadly between 20% and 70%, declines steadily with age, and is strongly influenced by previous calcium intake, the presence of other nutrients, pregnancy, lactation, overall calcium balance, and the availability of vitamin D.19–23 Fecal calcium includes the residual fraction of dietary calcium that is not absorbed, as well as a contribution from secreted calcium present in bile and other digestive juices (“endogenous fecal calcium”). Endogenous fecal calcium in humans normally amounts to 100 to 200 mg/d and is relatively unaffected by changes in dietary or serum calcium.^24,25

Intestinal calcium absorption is adjusted physiologically in response to variations in calcium intake, as shown by radiocalcium kinetic and balance studies in normal subjects.^26,27 Obligate renal and intestinal excretion of calcium is such that calcium balance cannot be maintained if dietary calcium consistently falls below 200 to 400 mg/d, even though the percentage of calcium absorbed may be very high (i.e., 70%). As calcium intake increases, overall calcium absorption (in mg/d) rises, but the fractional absorption of ingested calcium declines progressively such that within the physiologic range of calcium intake, total net calcium absorption tends to plateau at approximately 400 mg/d. Consequently, urinary calcium excretion tends also to plateau at higher intakes. Additional buffering of changes in dietary calcium results from control of renal tubular calcium reabsorption and skeletal calcium release, but regulation of intestinal absorptive efficiency is critical for calcium balance.²⁶ The mechanisms of this inverse regulation of intestinal absorptive efficiency by changes in calcium availability are considered in the following discussion.

Mechanisms and Sites of Calcium Absorption

Calcium is absorbed throughout the intestine. In terms of rates of transport per unit length of mucosa, absorption is most efficient in the duodenum and proximal jejunum, which exhibit the highest levels of vitamin D–dependent calcium binding proteins and in which lower luminal pH (5 to 6) promotes dissociation of calcium from complexes with food constituents and other ions.^28,29 On the other hand, longer residence times in the more distal small bowel segments may allow absorption of a larger proportion of total calcium intake in the distal jejunum and ileum.^30,31 The ileum, for example, may become an important site of net calcium absorption during dietary restriction of calcium or when the residence time of luminal contents in more proximal bowel segments is reduced.^30,32

Transcellular calcium absorption necessarily involves three steps: entry into the cell, diffusion across the cell, and extrusion from the cell (see Chapter 3). The first step, entry through the apical brush border, occurs via a member of the vanilloid (TRPV) superfamily of channels, namely TRPV6 (previously referred to as CaT1 or ECaC2).^33,34 Another member of this family, TRPV5, appears to be dominantly involved in calcium reabsorption in the kidney (see later). The TRPV6 and TRPV5 genes are located on chromosome 7q33-35. Their structure includes six transmembrane regions, a short hydrophobic region between segments 5 and 6 that likely forms the calcium pore, and large intracellular domains at the N- and C-termini.³³ The pore region is highly selective for calcium,^35,36 and mutations in this region have been shown to abolish calcium permeability.³⁷ Posttranslational modification and glycosylation of TRPV6 occurs, but the functional effects of these modifications are not well defined. Similarly, the mechanisms regulating the activity of these molecules are not well understood. TRPV6 and TRPV5 expression varies significantly between species and in humans, and both genes are expressed in a variety of tissues.³⁸ Specifically, TRPV5 seems to be the major isoform expressed in the kidney, whereas TRPV6 is most highly expressed in the small intestine (restricted to the apical surface of epithelial cells).³⁹ Given the important role of 1,25(OH)₂D₃ and other hormonal regulators in calcium absorption, the effects of these hormones on TRPV6 expression has recently been investigated. 1,25(OH)D₃ clearly increases TRPV6 expression in vitro (see Chapter 3).⁴⁰ Furthermore, mice with targeted inactivation of the vitamin D receptor demonstrate a 90% reduction in TRPV6 expression and a threefold reduction in intestinal calcium absorption.⁴¹ Additionally, calcium exposure appears to independently affect TRPV6 expression insofar as high calcium intake decreases TRPV6 expression, and low intake increases it.^41,42 Finally, TRPV6 expression is decreased in mice with targeted inactivation of their estrogen receptor-α gene and is increased in mice receiving exogenous estradiol administration.⁴³

After calcium has entered the cell, it must be transported through the cytoplasm and extruded from the cell against a steep gradient. The mechanism by which calcium moves through the cell involves the small cytosolic protein calbindin D_9k. Calcium entering the cell via the apical TRPV6 channel becomes tightly associated with the calbindin, which buffers the relatively large mass of entering calcium and minimizes its impact upon cytosolic free-calcium concentrations. The calbindin/calcium complex then diffuses across the cytosol to the basolateral membrane. Free calcium then dissociates into the low-cytosolic calcium environment maintained immediately subjacent to the basolateral membrane by high-affinity membrane Ca²⁺-ATPases located there. Finally, these calcium ATPases actively extrude calcium out of the cell.²⁹ The known cellular concentrations and kinetic properties of the calbindin D_9k molecule would support observed rates of duodenal calcium transport at submicromolar concentrations of cytosolic free calcium.⁴⁴ Indeed, the importance of this buffered diffusional process predicts that enterocyte calbindin D_9k content is likely to be a major determinant, along with TRPV6 activity, of the overall rate of enterocyte calcium transport.

Renal Calcium Excretion

Regulation of renal calcium excretion is an important mechanism for homeostatic control of blood ionized calcium in the face of fluctuations in filtered load, as derived from intestinal calcium absorption and net bone resorption. When urinary calcium is viewed as a function of the amount of calcium actually absorbed by the gut (i.e., after regulation of intestinal absorptive efficiency has been factored out), the precision with which the kidney adjusts tubular calcium reabsorption to residual changes in filtered load becomes obvious. Ordinarily, the daily load of calcium filtered at the glomerulus (the product of the glomerular filtration rate⁴⁵ and ultrafilterable calcium⁴⁶) is approximately 10,000 mg/d in adult humans, which means that the extracellular calcium pool is completely filtered several times a day. Since urinary calcium excretion (and net intestinal calcium absorption) is approximately 200 mg/d, only 2% of filtered calcium is excreted normally. This high ratio of filtered to excreted calcium affords ample opportunity for finely tuned hormonal control of calcium excretion, even though it may be difficult to measure the small changes involved.

Sites and Mechanisms of Renal Calcium Reabsorption

Calcium is reabsorbed at multiple sites and by different mechanisms along the nephron. As in the intestine, the challenge of transporting calcium across the renal epithelium requires that adequate rates of transport be achieved without substantially increasing the concentration of calcium within the cytosol of the epithelial cell. This is accomplished mainly by use of paracellular diffusional mechanisms that are supplemented in some nephron segments by active transcellular transport, especially in response to hormonal stimulation.

Approximately 60% to 70% of tubular calcium reabsorption, like that of sodium, occurs in the proximal tubule.⁴⁶ Proximal tubular calcium reabsorption occurs mainly via passive diffusion along paracellular pathways, down the ambient (lumen-positive) electrochemical gradient.⁴⁷ Another 20% to 25% of the filtered calcium load is reabsorbed in Henle’s loop.⁴⁶ This occurs mainly by paracellular diffusion in the cortical thick ascending limb (cTAL),^48–50 although active transcellular transport may play a minor role as well.^51,52 Calcium (and magnesium) reabsorption in this segment is severely impaired in patients with homozygous inactivating mutations in claudin 16 (CLDN16) (previously referred to as paracellin-1) or claudin 19 (CLDN19) protein, strongly expressed in tight junctions of the cTAL and presumably required for normal divalent cation conductance.^53–56 Hypercalcemia suppresses renal tubular calcium reabsorption even in the absence of PTH,⁵⁷ an effect likely due to direct activation of basolateral CaSRs in the cTAL (and possibly other nephron segments).⁵⁸ In the cTAL, activated CaSRs lower apical K⁺ exit by inhibiting renal outer medullary K⁺ channels (ROMKs), thereby blunting Na-K-2Cl cotransporter (NKCC2) activity and reducing the transepithelial voltage gradient that drives paracellular cation movement.^17,59–61 The critical importance of normal rates of Na-K-2Cl reabsorption for adequate calcium transport in the cTAL is highlighted clinically by the occurrence of dramatic hypercalciuria in patients with Bartter’s syndromes, which involve defects in NKCC2, ROMK, or other transporters or channels required for effective Na-K-2Cl reabsorption.⁶² An analogous Bartter’s-like phenotype may occur in some patients with activating CaSR mutations.⁶³ CaSR activation in the cTAL also antagonizes PTH-stimulated increases in calcium reabsorption, possibly by impairing cyclic adenosine monophosphate (cAMP) generation⁶¹ (see later).

Only about 8% to 10% of filtered calcium is reabsorbed in more distal segments of the tubule, but calcium transport in the distal nephron is a key control point for hormonal regulation and involves predominantly transcellular reabsorption.64–66 Cells in the late distal tubule exhibit striking co-localization of several unique proteins that are critical for effective transcellular calcium reabsorption. These include an apical epithelial calcium channel, TRPV5 (formerly ECaC1 or CaT2 and highly homologous to TRPV6, discussed earlier), the cytosolic calcium-binding protein, calbindin D_28k, basolateral plasma membrane calcium ATPase(s) (PMCAs), and basolateral NA⁺/Ca²⁺ exchanger 1 (NCX1).^67–72 As in the intestinal epithelium, these cells admit calcium across their apical membranes via opening of TRPV5. Expression of this channel is up-regulated by PTH, thus enhancing calcium reabsorption (see later). In contrast, high intracellular calcium concentrations and increased calbindin D_28k levels reduce calcium reabsorption, possibly via enhanced buffering of subapical Ca²⁺ ions. Calcium bound to calbindin D_28k is ferried across the cell to the basolateral membrane, where it ultimately is extruded against a steep electrochemical gradient, predominantly via NCX1 (70% of Ca²⁺ flux),^73,74 but also by PMCA. As expected, mice lacking TRPV5⁷⁵ or calbindin D_28k⁷⁶ were found to manifest severe hypercalciuria.

Additional calcium absorption may occur in more distal nephron segments, such as cortical and medullary collecting ducts.⁴⁷ Proteins other than those described above may be involved in renal calcium reabsorption as well, including voltage-operated calcium channels^47,77 and Cl⁻ channels, as evidenced by the hypercalciuria seen in Dent’s disease, an X-linked disorder due to inactivating mutations in the Cl⁻ channel ClC-5 that may be involved in endocytic vesicle function and protein trafficking.^78,79

Regulation of Renal Calcium Reabsorption

Calcium excretion is affected by a variety of hormones, ions, nutrients, and drugs.^80,81 Among these, PTH is the principal physiologic regulator of renal tubular calcium transport, as appreciated clinically by the markedly abnormal overall relation of serum to urinary calcium in hyper- and hypoparathyroidism.^80,82,83 PTH acts to enhance tubular calcium reabsorption in the cTAL, distal convoluted tubules (DCT), and connecting tubules (CNT).^50,65,84,85 It augments DCT calbindin D_28k expression⁸⁶ and basolateral N⁺/Ca²⁺ exchange,^87–91 increases the affinity for calcium of the basolateral Ca²⁺-ATPase,⁹² and hyperpolarizes the DCT cell,⁹³ which then activates the TRPV5 channel.⁹⁴ In immortalized murine distal tubular cells, PTH causes insertion into the apical membrane of new dihydropyridine-sensitive membrane calcium channels,⁷⁷ and it stimulates stretch-activated nonselective cation channels in rabbit CNT.⁹⁵ These are distinct from TRPV5 channels and may represent other mechanisms of apical calcium entry. PTH increases the surface expression or activity of TRPV5 epithelial calcium channels. In the cTAL, PTH also may increase some form of active transcellular transport,^51,52 and it may stimulate paracellular diffusional calcium transport by augmenting the transepithelial voltage gradient, at least in some segments and species.^96,97

Studies in patients with inactivating vitamin D receptor mutations have demonstrated that 1,25(OH)₂D₃ is required for the calcium-reabsorptive response to PTH.⁹⁸ It furthermore accelerates the increase in DCT cell calcium entry initiated by PTH,⁹⁹ and it directly increases DCT calcium reabsorption, apparently by driving higher expression of TRPV5, calbindin D_28k, and PMCA (see Chapter 3).^74,100–103 Another hormone affecting calcium reabsorption is calcitonin. When given in large doses, calcitonin acutely reduces proximal tubular calcium reabsorption by a mechanism independent of PTH;¹⁰⁴ however, an important role for calcitonin in the physiologic regulation of calcium reabsorption is thought to be unlikely. Hypercalciuria observed in states of excess growth hormone or cortisol seems likely to be secondary to an increased filtered load of calcium rather than to direct tubular actions of these hormones.^105–110 Estrogen treatment of normal postmenopausal women lowers urinary calcium excretion by increasing tubular calcium reabsorption.^111,112 In DCT of ovariectomized rats, 17β-estradiol acutely increases mRNA expression of all major components of the calcium transport pathway, including TRPV5, calbindin D_28k, NCX1, and PMCA1b.¹¹³ Reported effects on tubular calcium reabsorption of insulin,¹¹⁴ glucagon,⁹⁶ antidiuretic hormone,⁹⁶ and angiotensin II¹¹⁵ are of uncertain physiologic or clinical significance.

Parathyroid Tumors

Parathyroid tumors may occur as an isolated and sporadic endocrinopathy or as part of inherited tumor syndromes,¹³¹ such as the multiple endocrine neoplasias (MEN) or hereditary hyperparathyroidism with jaw tumors (HPT-JT),¹³² or in response to chronic overstimulation, as in uremic hyperparathyroidism.¹³³ Genetic analyses of kindreds with MEN1 and MEN2A and of tumor tissue from patients with single parathyroid adenomas have shown that some of the molecular mechanisms known to be involved in tumor genesis can also be responsible for the development of hyperparathyroidism.

Our current understanding indicates that sporadic parathyroid tumors are caused by single somatic mutations that lead to the activation or overexpression of proto-oncogenes such as parathyroid adenoma 1 (PRAD1) or RET (see Fig. 5-4A). Furthermore, different tumor-suppressor genes affecting the parathyroid glands are predicted to be located on several different chromosomes, and in a significant number of patients, LOH has been documented for one of these loci. For all these somatic mutations, a single point mutation or a deletion provides a growth advantage to a single parathyroid cell and its progeny, leading to their clonal expansion.

In hereditary forms of the disease, two distinct, sequentially occurring molecular defects are observed. The first “hit” (point mutation or deletion) is an inherited genetic defect, which affects only one allele that comprises a gene encoding an anti-oncogene (see Fig. 5-4B). Subsequently, a somatic mutation or deletion affecting the second allele occurs in a single parathyroid cell, and because of the resulting growth advantage, this mutation leads to its monoclonal expansion and thus the development of parathyroid tumors. Examples of this latter molecular mechanism in the development of hyperparathyroidism are the inactivation of tumor-suppressor genes such as the multiple endocrine neoplasia type 1 (MEN1) gene, the hyperparathyroidism–jaw tumor (HPT–JT) gene, and the retinoblastoma (Rb) gene (see Table 5-1).

Parathyroid Adenoma 1 and PTH Genes

Investigations of the PTH gene in sporadic parathyroid adenomas detected abnormally sized restriction fragment length polymorphisms (RFLPs) with a DNA probe for the 5′ part of the PTH gene in some adenomas,¹³⁴ indicating disruption of the gene. Further studies of the tumor DNA demonstrated that the first exon of the PTH gene (see Fig. 5-2) was separated from the fragments containing the second and third exons, and that a rearrangement had occurred, juxtaposing the 5′ PTH regulatory elements with “new” non-PTH DNA.¹³⁵ This rearrangement was not found in the DNA from the peripheral leukocytes of the patients, thereby indicating that it represented a somatic event and not an inherited germline mutation. Investigation of this rearranged DNA sequence localized it to chromosome 11q13, and detailed analysis revealed that it was highly conserved in different species and expressed in normal parathyroids and in parathyroid adenomas. The protein expressed as a result of this rearrangement, which was designated PRAD1, was demonstrated to encode a 295-amino-acid member of the cyclin-D family of cell-cycle regulatory proteins. Cyclins were initially characterized in the dividing cells of budding yeast, where they controlled the G₁ to S transition of the cell cycle, and in marine mollusks, where they regulated the mitotic phase (M-phase) of the cell cycle.¹³⁶ Cyclins have also been identified in man and have an important role in regulating many stages of cell-cycle progression. Thus PRAD1, which encoded a novel cyclin referred to as cyclin D1 (CCND1), is an important cell-cycle regulator, and overexpression of PRAD1 may be an important event in the development of at least 15% of sporadic parathyroid adenomas.^137,138

Interestingly, more than 66% of the transgenic mice overexpressing PRAD1 under the control of a mammary tissue-specific promoter were found to develop breast carcinoma in adult life,¹³⁹ and expression of this proto-oncogene under the control of the 5′ regulatory region of the PTH gene resulted in mild to moderate chronic hyperparathyroidism.^137,138 Taken together, these findings in transgenic animals provide further evidence for the conclusion that PRAD1 can be involved in the development of a significant number of parathyroid adenomas.

In addition to the rearrangement of the PTH gene in some parathyroid adenomas, a nonsense mutation of the PTH gene, Arg83Stop, that occurred in association with LOH of the PTH locus has been reported in a parathyroid adenoma.¹⁴⁰ The patient, who had presented with hypercalcemia and an undetectable serum PTH concentration, showed heterozygosity in the peripheral-blood leukocytes with wild-type and mutant alleles, but the parathyroid adenoma had a loss of the wild-type allele and retention of the mutant (Arg83Stop) allele, which predicts that the tumor secretes only a PTH peptide that is truncated after the 52nd amino acid. Following removal of the parathyroid adenoma, normocalcemia was restored.¹⁴⁰ These findings demonstrate that PTH nonsense mutations, which result in truncated forms of PTH not recognizable by standard hormone assays, may be associated with parathyroid adenoma, and that endogenously produced N-terminal PTH fragments can be biologically active.¹⁴⁰

Multiple Endocrine Neoplasia 1 Gene

MEN1 is characterized by the combined occurrence of tumors of the parathyroids, pancreatic islet cells, and anterior pituitary (Table 5-2).^141,142 Parathyroid tumors occur in 95% of MEN1 patients, and the resulting hypercalcemia is the first manifestation of MEN1 in about 90% of patients. Pancreatic islet cell tumors occur in 40% of MEN1 patients; gastrinomas, leading to the Zollinger-Ellison syndrome, are the most common type and also the important cause of morbidity and mortality in MEN1 patients. Anterior pituitary tumors occur in 30% of MEN1 patients, with prolactinomas representing the most common type. Associated tumors, which may also occur in MEN1, include adrenal cortical tumors, carcinoid tumors, lipomas, and cutaneous angiofibromas and collagenomas.^142,143 The gene causing MEN1 was localized to a less than 300-kb region on chromosome 11q13 by genetic mapping studies that investigated MEN1-associated tumors for LOH and by segregation studies in MEN1 families.¹⁴⁴ The results of these studies, which were consistent with Knudson’s model for tumor development, indicated that the MEN1 gene represented a putative tumor-suppressor gene (see Fig. 5-4B). Characterization of genes from this region led to the identification of the MEN1 gene, which consists of 10 exons that encode a novel 610-amino-acid protein referred to as menin.^145,146 Over 1100 germline MEN1 mutations have been identified, and the majority (>80%) are inactivating and are consistent with its role as a tumor-suppressor gene.^147,148 These mutations are diverse in their types, and in approximate percentages, 25% are nonsense, 45% are frameshift deletions or insertions, 9% are splice-site mutations, 20% are missense mutations, and 1% are whole or partial gene deletions.^{144,147–149} In addition, the MEN1 mutations are scattered throughout the 1830-bp coding region of the gene with no evidence for clustering. Correlations between the MEN1 germline mutations and the clinical manifestations of the disorder appear to be absent.^147–150 Tumors from MEN1 patients and non-MEN1 patients have been observed to harbor the germline mutation together with a somatic LOH involving chromosome 11q13, as expected from Knudson’s model and the proposed role of the MEN1 gene as a tumor suppressor.^{148,151–161} The role of the MEN1 gene in the etiology of familial isolated hyperparathyroidism (FIHP) has also been investigated, and germline MEN1 mutations have been reported in 29 families with FIHP.^{148,151,162–164} The sole occurrence of parathyroid tumors in these families is remarkable, and the mechanisms that determine the altered phenotypic expressions of these mutations remain to be elucidated.

The function of menin has been investigated by identifying its interactions with other proteins, and by under- or overexpression in in vitro studies. Menin has no homology to any known proteins or sequence motifs other than three nuclear localization signals (NLSs) in its C-terminal segment. Subcellular localization studies have shown that menin is predominantly a nuclear protein in nondividing cells, but in dividing cells, it is found in the cytoplasm. Menin has been shown to interact with a number of proteins that are involved in transcriptional regulation, genome stability, cell division, and proliferation.¹⁴⁸

The functional role of menin as a tumor suppressor also has been investigated, and studies in human fibroblasts have revealed that menin acts as a repressor of telomerase activity via hTERT (a protein component of telomerase).¹⁶⁵ Furthermore, overexpression of menin in the human endocrine pancreatic tumor cell line (BON1) resulted in an inhibition of cell growth¹⁶⁶ that was accompanied by up-regulation of JunD expression but down-regulation of delta-like protein 1/preadipocyte factor-1, proliferating cell nuclear antigen, and QM/Jif-1, which is a negative regulator of c-Jun.¹⁶⁶ These findings of growth suppression by menin were observed in other cell types. Thus, expression of menin in the RAS-transformed NIH3T3 cells partially suppressed the RAS-mediated tumor phenotype in vitro and in vivo.¹⁶⁷ Overexpression of menin in CHO-IR cells also suppressed insulin-induced AP-1 transactivation, and this was accompanied by an inhibition of c-Fos induction at the transcriptional level.¹⁶⁸ Furthermore, menin reexpression in Men1-deficient mouse Leydig tumor cell lines induced cell cycle arrest and apoptosis.¹⁶⁹ In contrast, depletion of menin in human fibroblasts resulted in their immortalization.¹⁶⁵ Thus, menin appears to have a large number of functions through interactions with proteins, and these mediate alterations in cell proliferation.

Multiple Endocrine Neoplasia 2 Gene (c-ret)

MEN2 describes the association (see Table 5-2) of medullary thyroid carcinoma (MTC), pheochromocytomas, and parathyroid tumors.^141,144 Three clinical variants of MEN2 are recognized: MEN2a, MEN2b, and MTC-only (see Table 5-2). MEN2a is the most common variant, and the development of MTC is associated with pheochromocytomas (50% of patients), which may be bilateral, and parathyroid tumors (20% of patients). MEN2b, which represents 5% of all MEN2 cases, is characterized by the occurrence of MTC and pheochromocytoma in association with a Marfanoid habitus, mucosal neuromas, medullated corneal fibers, and intestinal autonomic ganglion dysfunction leading to multiple diverticula and megacolon. Parathyroid tumors do not usually occur in MEN2b. MTC-only is a variant in which medullary thyroid carcinoma is the sole manifestation of the syndrome. The gene causing all three MEN2 variants was mapped to chromosome 10cen-10q11.2, a region containing the c-ret proto-oncogene which encodes a tyrosine kinase receptor with cadherin-like and cysteine-rich extracellular domains, and a tyrosine kinase intracellular domain.^170,171 Specific mutations of c-ret have been identified for each of the three MEN2 variants. Thus in 95% of patients, MEN2a is associated with mutations of the cysteine-rich extracellular domain, and mutations in codon 634 (Cys→Arg) account for 85% of MEN2A mutations. However, a search for c-ret mutations in sporadic non-MEN2a parathyroid adenomas revealed no codon 634 mutations.^172,173 MTC-only is also associated with missense mutations in the cysteine-rich extracellular domain, and most mutations are in codon 618. However, MEN2b is associated with mutations in codon 918 (Met→Thr) of the intracellular tyrosine kinase domain in 95% of patients. Interestingly, the c-ret proto-oncogene is also involved in the etiology of papillary thyroid carcinomas and in Hirschsprung’s disease. Mutational analysis of c-ret to detect mutations in codons 609, 611, 618, 634, 768, and 804 in MEN2a and MTC-only, and codon 918 in MEN2b, has been used in the diagnosis and management of patients and families with these disorders.^171,174

Hyperparathyroidism–Jaw Tumor Syndrome Gene

The HPT–JT syndrome is an autosomal-dominant disorder characterized by the development of parathyroid adenomas and carcinomas, and fibro-osseous jaw tumors.^175,176 In addition, some patients may also develop uterine tumors and renal abnormalities, which include Wilms’ tumors, renal cysts, renal hamartomas, renal cortical adenomas, and papillary renal cell carcinomas.¹⁷⁷ Other tumors, including pancreatic adenocarcinomas, testicular mixed germ cell tumors with a major seminoma component, and Hurthle cell thyroid adenomas have also been reported in some patients.^132,177 It is important to note that the parathyroid tumors may occur in isolation and without any evidence of jaw tumors, and this may cause confusion with other hereditary hypercalcemic disorders such as MEN1, familial benign hypercalcemia (FBH) (which is also referred to as familial hypocalciuric hypercalcemia [FHH]), and FIHP.¹⁷⁸ HPT–JT can be distinguished from FBH, because in FBH, serum calcium levels are elevated during the early neonatal or infantile period, whereas in HPT–JT, such elevations are uncommon in the first decade. In addition, HPT–JT patients, unlike FBH patients, will have associated hypercalciuria. The distinction between HPT–JT patients and MEN1 patients, who have only developed the usual first manifestation of hypercalcemia (>90% of patients), is more difficult and is likely to be influenced by operative and histologic findings and by the occurrence of other characteristic lesions in each disorder. It is important to note that HPT–JT patients will usually have single adenomas or a carcinoma, but MEN1 patients will often have multiglandular parathyroid disease. The distinction between FIHP and HPT–JT in the absence of jaw tumors is difficult but important; HPT–JT patients may be at a higher risk of developing parathyroid carcinomas.^179–181 These distinctions may be helped by the identification of additional features, and a search for jaw tumors, renal, pancreatic, thyroid, and testicular abnormalities may help to identify HPT–JT patients. The jaw tumors in HPT–JT are different from the brown tumors observed in some patients with primary hyperparathyroidism and do not resolve after parathyroidectomy.¹⁷⁸ Indeed, ossifying fibromas of the jaw are an important distinguishing feature of HPT–JT from FIHP, and the occurrence of these may occasionally precede the development of hypercalcemia in HPT–JT patients by several decades. The gene causing HPT–JT is located on chromosome 1q31.2 and consists of 17 exons that encode a ubiquitously expressed and evolutionarily conserved 531-amino-acid protein, designated parafibromin.^132,182 This gene, CDC73, is also referred to as HRPT2 (i.e., hyperparathyroidism type 2). HRPT2 mutations associated with HPT–JT were found to be scattered throughout the 1593-bp coding region, with the majority (>80%) predicting a functional loss through premature truncation. A genotype-phenotype correlation was not apparent from these analyses.^177,182,184 The observation of LOH involving the chromosome lq31.2 region in HPT–JT-associated tumors indicated that parafibromin may be acting as a tumor suppressor, consistent with Knudson’s two-hit hypothesis.^177,178,182 This was supported by the observations of germline and somatic HRPT2 mutations in HPT–JT-associated tumors.^{177,182–185} Similar germline and somatic HRPT2 mutations have also been found in sporadic parathyroid carcinomas, and the frequency of such mutations is high, ranging from 67% to 100%^183,186; however, the frequency of HRPT2 mutations in sporadic parathyroid adenomas is low at 0% to 4%, indicating that HRPT2 mutations likely confer an aggressive growth potential to the parathyroid cells.^{182–184,186,187} HRPT2 mutations and allelic imbalances have also been identified in sporadic renal tumors,¹⁸⁸ and a loss or down-regulation of HRPT2 expression has been reported in both breast and gastric cancers.^189,190 These studies indicate that HRPT2 and its encoded protein, parafibromin, play a critical role in inherited and sporadic parathyroid cancers as well as other nonhereditary solid tumors. The role of parafibromin, which is predominantly a nuclear protein with a monopartite NLS,¹⁹¹ was not readily apparent, inasmuch as it has no homologies to known proteins. However, the approximately 200 amino acids of the C-terminal domain shared over 25% sequence identity with the yeast Cdc73 protein which is a component of the yeast polymerase-associated factor 1 (PAF1) complex, a key transcriptional regulatory complex that interacts directly with RNA polymerase II.^182,192,193 Studies of the PAF1 complex in yeast and Drosophila, as well as in mammalian cells, have revealed that parafibromin, as part of the PAF1 complex, is a mediator of the key transcriptional events of histone modification, chromatin remodeling, initiation and elongation, and the wnt/β-catenin signaling pathway.^192–194 Studies of a mouse deleted for HRPTt2 have revealed that HRPT2 expression and the PARAFIBROMIN/PAF complex directly regulate genes (e.g., H19, IgF1, Igf3, Igfbp4, Hmga1, Hmga2, and Hmga3) that are involved in cell growth and apoptosis.¹⁹⁵

LRP5 And The Wnt/β-Catenin Pathway

Aberrant Wnt/β-catenin signaling with an accumulation of β-catenin in the cytoplasm and nucleus is associated with several types of tumor development (e.g., adenomatous polyposis coli and colorectal cancer). Investigations of this pathway in parathyroid tumors have revealed that β-catenin accumulation occurs in parathyroid adenomas and in parathyroid tumors associated with chronic renal failure.¹⁹⁶ In addition, a protein-stabilizing mutation, Ser37Ala, in exon 3 of β-catenin was detected in over 7% of parathyroid adenomas but not parathyroid tumors of chronic renal failure, from Swedish patients^197,198 but not North American¹⁹⁹ or Japanese²⁰⁰ patients. The Ser37Ala β-catenin mutations were homozygous in the parathyroid adenomas, which had a higher expression of β-catenin and the nonphosphorylated active form of β-catenin.¹⁹⁸ In addition, MYC, which is a direct target of the Wnt/β-catenin signaling pathway in colorectal cancer cells and a critical mediator of the early stages of intestinal neoplasia, was also overexpressed, and the stable activity of endogenous β–catenin was found to be necessary for MYC and cyclin D1 expression.¹⁹⁶ Stability of β-catenin is regulated by Wnt ligands, which bind to the cell-surface frizzled receptors and LRP5 and LRP6 co-receptors that alter phosphorylation of several intracellular second messengers and consequently accumulation of nonphosphorylated β-catenin. Investigation of the Wnt signaling pathway in parathyroid tumors revealed that over 85% of adenomas and 100% of tumors from chronic renal failure patients have a shorter LRP5 transcript, which contained an in-frame deletion of 142 amino acids (residues 666 to 809) that encompassed the third YWTD beta propeller domain between the second and third epidermal growth factor repeats.¹⁹⁷ This internally truncated LRP5 receptor activated β-catenin signaling in parathyroid tumors by a mechanism that may involve an impaired inhibitory action of the WNT antagonist, DKK1.¹⁹⁷ The parathyroid tumors expressing the internally truncated LRP5 receptor did not harbor the β-catenin stabilizing mutation, Ser37Ala, and those that had the stabilizing β-catenin mutation did not express the truncated LRP5 receptor.¹⁹⁷ Thus, it seems that the presence of the stabilizing β-catenin mutation and the expression of the truncated LRP5 receptor are mutually exclusive. However, these studies demonstrate an important role for the WNT β-catenin signaling pathway in parathyroid tumorigenesis.

Rb Gene

The Rb gene, which is a tumor-suppressor gene²⁰¹ located on chromosome 13q14, is involved in the pathogenesis of retinoblastomas and a variety of common sporadic human malignancies including ductal breast, small cell lung carcinoma, and bladder carcinomas. Allelic deletion of the Rb gene has been demonstrated in all parathyroid carcinomas and in 10% of parathyroid adenomas^202,203 and was accompanied by abnormal staining patterns for the Rb protein in 50% of the parathyroid carcinomas but in none of the parathyroid adenomas.²⁰² These results demonstrate an important role for the Rb gene in the development of parathyroid carcinomas and may be of help in the histologic distinction of parathyroid adenoma from carcinoma.²⁰² The Rb protein may also be secondarily involved in parathyroid tumorigenesis by its interaction with the retinoblastoma-interacting zinc finger protein 1, RIZ1.²⁰⁴ The findings of extensive deletions of the long arm of chromosome 13 (including the Rb locus) in some parathyroid adenomas and carcinomas,²⁰³ and similar findings in pituitary carcinomas,²⁰⁵ suggest that other tumor-suppressor genes on chromosome 13q may also have a role in the development of such tumors.

Retinoblastoma-Interacting Zinc Finger Protein 1 Gene On Chromosome 1P

Loss of heterozygosity studies have revealed allelic loss of chromosome 1p32-pter in 40% of sporadic parathyroid adenomas.²⁰⁶ This region is estimated to be about 110 cM, equivalent to about 110 million base pairs (Mbp) of DNA, but additional studies narrowed the interval containing this putative tumor-suppressor gene(s) to an approximately 4-cM (i.e., about 4-Mbp) region.²⁰⁷ Investigations of one candidate gene, retinoblastoma-interacting zinc finger protein 1 (RIZ1), have revealed that over 25% of parathyroid tumors had LOH of the RIZ1 locus, and that over 35% of parathyroid tumors had hypermethylation of the RIZ1 promoter region.²⁰⁴ Moreover, the RIZ1 promoter hypermethylation was related to LOH in these tumors, indicating that these two events may represent the “two hits” (see Fig. 5-4B) required for tumor development in Knudson’s hypothesis for tumorigenesis.²⁰⁴

Nonsyndromic Familial Isolated Hyperparathyroidism

FIHP may represent an incomplete manifestation of a syndromic form such as MEN1, FHH, or HPT-JT.163,164,182,208 Twenty-nine MEN1, nine HRPT2 and five CaSR germline mutations have been reported to date in FIHP kindreds.¹⁶⁴ However, it is important to note that the genetic etiology of nonsyndromic FIHP in the majority of families remains to be elucidated.^209,210 Thus, studies of 32 kindreds with nonsyndromic FIHP for mutations of the MEN1, CaSR, and HRPT2 genes have revealed that only one family harbored a germline mutation, and this involved the HRPT2 gene that encodes parafibromin.^209,210 However, studies of 10 other FIHP kindreds have indicated that another locus, referred to as HRPT3, on chromosome 2p13.3-p14, is likely to be involved in the etiology of nonsyndromic FIHP.²¹¹ Thus, the genes and their underlying abnormalities that lead to nonsyndromic FIHP remain to be identified.

Hyperparathyroidism In Chronic Renal Failure

Chronic renal failure is often associated with a form of secondary hyperparathyroidism that may subsequently result in the hypercalcemic state of “tertiary” hyperparathyroidism. The parathyroid proliferative response in this condition led to the proposal that the autonomous parathyroid tissue might have undergone hyperplastic change and therefore be polyclonal in origin. However, studies of X-chromosome inactivation in parathyroids from patients on hemodialysis with refractory hyperparathyroidism have revealed at least one monoclonal parathyroid tumor in over 60% of patients.¹³³ In addition, LOH involving several loci on chromosome Xp11 was detected in one of these parathyroid tumors, thereby suggesting the involvement of a tumor-suppressor gene from this region in the pathogenesis of such tumors.¹³³ Interestingly, none of the parathyroid tumors from these patients with chronic renal failure had LOH involving loci from chromosome 11q13. This unexpected finding of monoclonal parathyroid tumors in the majority of patients with “tertiary” hyperparathyroidism suggests that an increased turnover of parathyroid cells in secondary hyperparathyroidism may possibly render the parathyroid glands more susceptible to mitotic nondisjunction or other mechanisms of somatic deletions, which may involve loci other than those—MEN1 and PRAD1—located on chromosome 11q13. In addition, as noted above, parathyroid tumors from patients with chronic renal failure have been shown to accumulate β-catenin and to have a truncated form of the LRP5 receptor, which lacks 142 amino acids.^196,197

Disorders of The Calcium-Sensing Receptor

Three hypercalcemic disorders due to mutations and/or reduced activity of CaSR have been reported,208,212–217 which are FBH, also referred to as FHH, neonatal severe hyperparathyroidism (NSHPT), and autoimmune hypocalciuric hypercalcemia (AHH) (Table 5-3).

Familial Benign Hypercalcemia and Neonatal Severe Hyperparathyroidism

Mutational analyses of the human CaSR, which is a G protein–coupled receptor located on chromosome 3q21.1,²¹⁸ have revealed different mutations that result in a loss-of-function of the CaSR in patients with FBH and NSHPT.^212–217 Many of these mutations cluster around the aspartate- and glutamate-rich regions (codons 39-300) within the extracellular domain of the receptor, and this has been proposed to contain low-affinity calcium-binding sites, based on similarities to that of calsequestrin, in which the ligand-binding pockets also contain negatively charged amino acid residues.^208,219 Approximately two-thirds of the FBH kindreds investigated have been found to have unique heterozygous mutations of the CaSR, and expression studies of these mutations have demonstrated a loss of CaSR function whereby there is an increase in the calcium ion–dependent set-point for PTH release from the parathyroid cell.^{212,217,220,221} NSHPT occurring in the offspring of consanguineous FBH families has been shown to be due to homozygous CaSR mutations.^{212,213,215,222,223} However, some patients with sporadic neonatal hyperparathyroidism have been reported to be associated with de novo heterozygous CaSR mutations,²¹⁴ thereby suggesting that factors other than mutant gene dosage²²²—for example, the degree of set-point abnormality, the bony sensitivity to PTH, and the maternal extracellular calcium concentration—may also all play a role in the phenotypic expression of a CaSR mutation in the neonate. The remaining third of FBH families in whom a mutation within the coding region of the CaSR has not been demonstrated may either have an abnormality in the promoter of the gene or a mutation at one of the two other FBH loci that have been revealed by family linkage studies. One of these FBH loci is located on chromosome 19p and is referred to as FBH_19p.²²⁴ Studies of another FBH kindred from Oklahoma that also suffered from progressive elevations in PTH, hypophosphatemia, and osteomalacia^225,226 demonstrated that this variant, designated FBH_Ok, was linked to chromosome 19q13.²²⁷ These three FBH loci located on chromosomes 3q, 19p, and 19q have also been referred to as FBH (or FHH) types 1, 2, and 3, respectively.²²⁷

Autoimmune Hypocalciuric Hypercalcemia

Some patients who have the clinical features of FHH but not CaSR mutations may have AHH (see also Chapter 7). Four patients with AHH who all had other autoimmune manifestations have been reported²²⁸: three patients had antithyroid antibodies, and one had sprue with antigliadin and antiendomysial antibodies. These patients were shown to have circulating antibodies to the extracellular domain of the CaSR, and these antibodies stimulated PTH release from dispersed human parathyroid cells in vitro, probably by inhibiting the activation of the CaSR by extracellular calcium.²²⁸ Thus, AHH is a condition of extracellular calcium sensing that should be considered in FHH patients who do not have CaSR mutations.

Jansen’s Disease

Jansen’s disease (Figs. 5-5 and 5-6) is an autosomal-dominant disease that is characterized by short-limbed dwarfism caused by an abnormal regulation of chondrocyte proliferation and differentiation in the metaphyseal growth plate and associated, usually severe, hypercalcemia and hypophosphatemia despite normal or undetectable serum levels of PTH or PTHrP.²²⁹ These abnormalities are caused by mutations in the PTH/PTHrP receptor that lead to constitutive, PTH- and PTHrP-independent receptor activation.^230–232 Three different heterozygous mutations of the PTH/PTHrP receptor have been identified in the severe form of Jansen’s disease, and these involve codon 223 (His→Arg), codon 410 (Thr→Pro), and codon 458 (Ile→Arg) (Fig. 5-7). Expression of the mutant receptors in COS-7 cells result in constitutive, ligand-independent accumulation of cAMP, while the basal accumulation of inositol phosphates is not measurably increased.^230–232 Since the PTH/PTHrP receptor is most abundantly expressed in kidney and bone and in the metaphyseal growth plate, these findings provide a likely explanation for the abnormalities observed in mineral homeostasis and growth plate development associated with this disorder. This conclusion is supported further by observations in mice which express the human PTH/PTHrP receptor, with the His223Arg mutation under the control of the rat α₁(II) promoter.²³³ This promoter targeted expression of the mutant receptor to the layer of proliferative chondrocytes, delayed their differentiation into hypertrophic cells, and led, at least in animals with multiple copies of the transgene, to a mild impairment in growth of long bones. These observations are consistent with the conclusion that expression of a constitutively active human PTH/PTHrP receptor in growth plate chondrocytes causes the characteristic metaphyseal changes in patients with Jansen’s disease.

Another novel heterozygous PTH/PTHrP receptor mutation, T410R, was identified in several members of a small kindred with an apparently mild form of Jansen’s disease.²³⁴ Affected individuals had, compared to patients with the previously identified activating PTH/PTHrP receptor mutations,^230–232 less severe growth plate abnormalities, relatively normal stature, normal plasma calcium concentration, yet significant hypercalciuria and normal or suppressed plasma PTH levels. When tested in vitro, the PTH/PTHrP receptor with the T410R mutation showed less constitutive activity than that observed with the previously described T410P mutant.^231,235 This less pronounced agonist-independent cAMP accumulation induced by the T410R mutation is consistent with the less severe skeletal and laboratory abnormalities observed in this milder form of Jansen’s disease.

Williams Syndrome

Williams syndrome is an autosomal-dominant disorder characterized by supravalvular aortic stenosis, elfin-like facies, psychomotor retardation, and infantile hypercalcemia. The underlying abnormality of calcium metabolism remains unknown, but abnormal 1,25(OH)₂D₃ metabolism or decreased calcitonin production have been implicated, although none have been consistently demonstrated. Studies have demonstrated hemizygosity at the elastin locus on chromosome 7q11.23 in over 90% of patients with the classical Williams phenotype,236–238 and only one patient had a cytogenetically identifiable deletion, thereby indicating that the syndrome is usually due to a microdeletion of 7q11.23.²³⁸ Interestingly, ablation of the elastin gene in mice results in vascular abnormalities similar to those observed in patients with Williams syndrome.²³⁹ However, the microdeletions that have been reported involve also another gene, designated LIM-kinase, that is expressed in the central nervous system.²⁴⁰ The calcitonin receptor gene, which is located on chromosome 7q21, is not involved in the deletions found in Williams syndrome and is therefore unlikely to be implicated in the hypercalcemia of such children.²⁴¹ While deletion of the elastin and LIM-kinase genes can explain the respective cardiovascular and neurologic features of Williams syndrome, it seems likely that another, as yet uncharacterized, gene that is within this contiguously deleted region is involved in this disorder and could explain the abnormalities of calcium metabolism.

Hypoparathyroidism

Hypoparathyroidism may occur as part of a pluriglandular autoimmune disorder or as a complex congenital defect, as for example in the DiGeorge syndrome. In addition, hypoparathyroidism may develop as a solitary endocrinopathy, and this has been called isolated or idiopathic hypoparathyroidism. Familial occurrences of isolated hypoparathyroidism with autosomal-dominant, autosomal-recessive, and X-linked-recessive inheritances have been established.

Parathyroid Hormone Gene Abnormalities

DNA sequence analysis of the PTH gene (see Fig. 5-2) from one patient with autosomal-dominant isolated hypoparathyroidism has revealed a single base substitution (T→C) in exon 2,²⁴² which resulted in the substitution of arginine (CGT) for cysteine (TGT) in the signal peptide. The presence of this charged amino acid in the midst of the hydrophobic core of the signal peptide impeded the processing of the mutant preproPTH, as demonstrated by in vitro studies. These revealed that the mutation impaired the interaction of the nascent protein with the translocation machinery and that cleavage of the mutant signal sequence by solubilized signal peptidase was ineffective.^242,243 Studies using transfected HEK293 cells showed that the mutant PTH is trapped intracellularly, predominantly in the endoplasmic reticulum (ER), which is toxic for the cells and leads to apoptosis.²⁴⁴ In another family with autosomal-recessive hypoparathyroidism, a single base substitution (T→C) involving codon 23 of exon 2 was detected. This resulted in the substitution of proline (CCG) for the normal serine (TCG) in the signal peptide.²⁴⁵ This mutation alters the −3 position of the pre-pro-PTH protein cleavage site. Indeed, amino acid residues at the −3 and −1 positions of the signal peptidase recognition site have to conform to certain criteria for correct processing through the rough endoplasmic reticulum (RER), and one of these is an absence of proline in the region −3 and +1 of the site. Thus, the presence of a proline, which is a strong helix-breaking residue, at the −3 position is likely to disrupt cleavage of the mutant pre-pro-PTH that would be subsequently degraded in the RER, and PTH would not be available.²⁴⁵ Another abnormality of the PTH gene, involving a donor splice site at the exon 2/intron 2 boundary, has been identified in one family with autosomal-recessive isolated hypoparathyroidism.²⁴⁶ This mutation involved a single base transition (g→c) at position 1 of intron 2, and an assessment of the effects of this alteration in the invariant gt dinucleotide of the 5′ donor splice site consensus on mRNA processing revealed that the mutation resulted in exon skipping, in which exon 2 of the PTH gene was lost, and exon 1 was spliced to exon 3. The lack of exon 2 would lead to a loss of the initiation codon (ATG) and the signal peptide sequence (see Fig. 5-2), which are required for the commencement of PTH mRNA translation and the translocation of the PTH peptide, respectively.

Glial Cells Missing B Abnormalities

Glial cells missing B (GCMB), which is the human homolog of the Drosophila gene, Gcm, and of the mouse gcm2 gene, is expressed exclusively in the parathyroid glands, suggesting that it may be a specific regulator of parathyroid gland development.²⁴⁷ Mice that were homozygous (−/−) for deletion of gcm2 lacked parathyroid glands and developed the hypocalcemia and hyperphosphatemia observed in hypoparathyroidism.²⁴⁷ However, despite their lack of parathyroid glands, Gcm2-deficient (−/−) mice did not have undetectable serum PTH levels, but instead had levels indistinguishable from those of eucalcemic normal (+/+, wild-type) and heterozygous (+/−) mice. This endogenous level of PTH in the Gcm2-deficient (−/−) mice was too low to correct the hypocalcemia, but exogenous continuous PTH infusion could correct the hypocalcemia.²⁴⁷ Interestingly, there were no compensatory increases in PTHrP or 1,25(OH)₂D₃. These findings indicate that Gcm2 mice have a normal response (and not resistance) to PTH, and that the PTH in the serum of Gcm2-deficient mice was active. The auxiliary source of PTH was identified to be a cluster of PTH-expressing cells under the thymic capsule. These thymic PTH-producing cells also expressed the CaSR, and long-term treatment of the Gcm2-deficient mice with 1,25(OH)₂D₃ restored the serum calcium concentrations to normal and reduced the serum PTH levels, thereby indicating that the thymic production of PTH can be down-regulated.²⁴⁷ However, it appears that this thymic production of PTH cannot be up-regulated, because serum PTH levels are not high despite the hypocalcaemia in the Gcm2-deficient mice. This absence of up-regulation would be consistent with the very small size of the thymic PTH-producing cell cluster when compared to the size of normal parathyroid glands. The development of the thymic PTH-producing cells likely involves Gcm1, which is the other mouse homolog of Drosophila Gcm.²⁴⁸ Gcm1 expression, which could not be detected in parathyroid glands, colocalized with PTH expression in the thymus.²⁴⁷ The specific role of Gcm2 in the development of the parathyroids from the third pharyngeal pouch has been further investigated by studying the expression of the Hoxa3-Pax1/9-Eya1 transcription factor and Sonic hedgehog–bone morphogenetic protein 4 (Shh-Bmp4) signaling networks.²⁴⁹ These studies have revealed that Gcm2 (−/−) embryos that are 12 d.p.c. have a parathyroid-specific domain, but that this parathyroid domain undergoes coordinated programmed cell death (apoptosis) by 12.5 d.p.c. in the Gcm2-null mouse embryos.²⁴⁹ Moreover, the expression of the transcription factors Hoxa3, Pax1, Pax9, Eya1, Tbx1, and of Shh and Bmp4 was normal in the third pharyngeal pouch of these Gcm2-null mouse embryos. These findings indicate that the Hoxa3-Pax1/9-Eya transcription factor cascade, the transcription factor Tbx1, and the Shh-Bmp4 signaling network all act upstream of Gcm2.²⁴⁹ Moreover, these studies have revealed that Gcm2 has a role in promoting differentiation and survival of parathyroid cells in the developing embryo.²⁴⁹

Studies of patients with isolated hypoparathyroidism have shown that GCMB mutations are associated with autosomal-recessive and autosomal-dominant forms of the disease.250–252 A homozygous intragenic deletion of GCMB has been identified in a patient with autosomal-recessive hypoparathyroidism,²⁵⁰ while in another family, a homozygous missense mutation (Arg47Leu) of the DNA binding domain has been reported.²⁵¹ Functional analysis, using electrophoretic mobility shift assays (EMSAs), of this Arg47Leu GCMB mutation revealed that it resulted in a loss of DNA binding to the GCM DNA binding site.²⁵¹ More recently, heterozygous GCMB mutations, which consist of single nucleotide deletions (c1389deT and c1399delC) that introduce frameshifts and premature truncations, have been identified in two unrelated families with autosomal-dominant hypoparathyroidism (Fig. 5-8).²⁵² By using a GCMB-associated luciferase reporter, both of these mutations were shown to inhibit the action of the wild-type transcription factor, thereby indicating that these GCMB mutants have dominant-negative properties.²⁵²

X-Linked Recessive Hypoparathyroidism

X-linked recessive hypoparathyroidism has been reported in two multigenerational kindreds from Missouri.^253,254 In this disorder, only males are affected, and they suffer from infantile onset of epilepsy and hypocalcemia due to an isolated defect in parathyroid gland development.²⁵⁵ Relatedness of the two kindreds has been established by demonstrating an identical mitochondrial DNA sequence, inherited via the maternal lineage, in affected males from the two families.²⁵⁶ Studies utilizing X-linked polymorphic markers in these families localized the mutant gene to chromosome Xq26-q27,²⁵⁷ and a molecular deletion-insertion that involves chromosome 2p25 and Xq27 has been identified.²⁵⁸ This deletion-insertion is located approximately 67 kb downstream of SOX3, and hence it is likely to exert a position effect on SOX3 expression. Moreover, SOX3 was shown to be expressed in the developing parathyroids of mouse embryos, and this indicates a likely role for SOX3 in the embryonic development of the parathyroid glands.²⁵⁸ SOX3 belongs to a family of genes encoding high-mobility group (HMG) box transcription factors and is related to SRY, the sex-determining gene on the Y chromosome. The mouse homolog is expressed in the prestreak embryo and subsequently in the developing central nervous system (CNS) that includes the region of the ventral diencephalon, which induces development of the anterior pituitary and gives rise to the hypothalamus, olfactory placodes, and parathyroids.^258–261 The location of the deletion-insertion approximately 67 kb downstream of SOX3 in X-linked recessive hypoparathyroid patients is likely to result in altered SOX3 expression, since SOX3 expression has been reported to be sensitive to position effects caused by X-chromosome abnormalities.²⁶² Indeed, reporter construct studies of the mouse Sox3 gene have demonstrated the presence of both 5′ and 3′ regulatory elements,²⁶³ and thus it is possible that the deletion-insertion in the X-linked recessive hypoparathyroid patients may have a position effect on SOX3 expression and parathyroid development from the pharyngeal pouches. Indeed such position effects on SOX genes, which may be exerted over large distances, have been reported. For example, the very closely related Sox2 gene has been shown to have regulatory regions spread over a long distance, both 5′ and 3′ to the coding region,²⁶⁴ and disruption of sequences at some distance 3′ have been reported to lead to loss of expression in the developing inner ear and absence of sensory cells, whereas expression in other sites is unaffected.²⁶⁵ Similarly for the SRY gene, which probably originated from SOX3,²⁶⁶ both 5′ and 3′ deletions result in abnormalities of sexual development, and translocation breakpoints over 1 Mb upstream of the SOX9 gene have been reported to result in Campomelic dysplasia due to removal of elements that regulate SOX9 expression.²⁶² The molecular deletion-insertion identified in X-linked recessive hypoparathyroidism may similarly cause position effects on SOX3 expression, and this points to a potential role for the SOX3 gene in the embryologic development of the parathyroid glands from the pharyngeal pouches.

Pluriglandular Autoimmune Hypoparathyroidism

Hypoparathyroidism may occur in association with candidiasis and autoimmune Addison’s disease, and the disorder has been referred to as either the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome or the autoimmune polyglandular syndrome type 1 (APS1).²⁶⁷ This disorder has a high incidence in Finland, and a genetic analysis of Finnish families indicated autosomal-recessive inheritance of the disorder.²⁶⁸ In addition, the disorder has been reported to have a high incidence among Iranian Jews, although the occurrence of candidiasis was less common in this population.²⁶⁹ Linkage studies of Finnish families mapped the APECED gene to chromosome 21q22.3.²⁷⁰ Further positional cloning approaches led to the isolation of a novel gene from chromosome 21q22.3. This gene, referred to as AIRE (autoimmune regulator), encodes a 545-amino-acid protein that contains motifs suggestive of a transcriptional factor and includes two zinc finger motifs, a proline-rich region, and three LXXLL motifs.²⁷¹ Four AIRE1 mutations are commonly found in APECED families: (1) Arg257 Stop in Finnish, German, Swiss, British, and Northern Italian families; (2) Arg139 Stop in Sardinian families; (3) Tyr85Cys in Iranian Jewish families; and (4) a 13-bp deletion in exon 8 in British, Dutch, German, and Finnish families.^271–275 AIRE1 has been shown to regulate the elimination of organ-specific T cells in the thymus, and thus APECED is likely to be caused by a failure of this specialized mechanism for deleting forbidden T cells and establishing immunologic tolerance.²⁷⁶ Patients with APS1 may also develop other autoimmune disorders in association with organ-specific autoantibodies, which are similar to those in patients with non-APS1 forms of the disease. Examples of such autoantibodies and related diseases are GAD6S autoantibodies in diabetes mellitus type 1A and 21-hydroxylase autoantibodies in Addison’s disease. Patients with APS1 may also develop autoantibodies that react with specific autoantigens that are not found in non-APS1 patients. Examples of this are autoantibodies to type 1 interferon omega (IFN-ω), which are present in all APS1 patients,²⁷⁷ and to NACHT leucine-rich-repeat protein 5 (NALP5), which is a parathyroid-specific autoantibody present in 49% of patients with APS1 associated hypoparathyroidism.²⁷⁸ NALP proteins are essential components of the inflammasome and activate the innate immune system in different inflammatory and autoimmune disorders such as vitiligo, which involves NALP1, and gout, which involves NALP3.²⁷⁹ The precise role of NALP5 in APS1-associated hypoparathyroidism remains to be elucidated.

Digeorge Syndrome

Patients with the DiGeorge syndrome (DGS) typically suffer from hypoparathyroidism; immunodeficiency; congenital heart defects; and deformities of the ear, nose, and mouth. The disorder arises from a congenital failure in the development of the derivatives of the third and fourth pharyngeal pouches, with resulting absence or hypoplasia of the parathyroids and thymus. Most cases of DGS are sporadic, but an autosomal-dominant inheritance of DGS has been observed. An association between the syndrome and an unbalanced translocation and deletions involving 22q11.2 have also been reported,²⁸⁰ and this is referred to as DGS type 1 (DGS1). In some patients, deletions of another locus on chromosome 10p have been observed in association with DGS,²⁸¹ and this is referred to as DGS type 2 (DGS2). Mapping studies of the DGS1 deleted region on chromosome 22q11.2 have defined a 250-kb to 3000-kb critical region^282,283 that contained approximately 30 genes. Studies of DGS1 patients have reported deletions of several of the genes (e.g., rnex40, nex2.2–nex3, UDF1L, and TBX1) from the critical region,^{280,284–286} and studies of transgenic mice deleted for such genes (e.g., Udf1l, Hira, and Tbx1) have revealed developmental abnormalities of the pharyngeal arches.^287–289 However, point mutations in DGS1 patients have only been detected in the TBX1 gene,²⁹⁰ and TBX1 is now considered to be the gene causing DGS1.²⁹¹ TBX1 is a DNA-binding transcriptional factor of the T-Box family and known to have an important role in vertebrate and invertebrate organogenesis and pattern formation. The TBX1 gene is deleted in roughly 96% of all DGS1 patients. Moreover, DNA sequence analysis of unrelated DGS1 patients who did not have deletions of chromosome 22q11.2 revealed the occurrence of three heterozygous point mutations.²⁹⁰ One of these mutations resulted in a frameshift with a premature truncation; the other two were missense mutations (Phe148Tyr and Gly310Ser). All of these patients had the complete pharyngeal phenotype but did not have mental retardation or learning difficulties. Interestingly, transgenic mice with deletion of Tbx1 have a phenotype that is similar to that of DGS1 patients.²⁸⁹ Thus, Tbx1-null mutant mice (−/−) had all the developmental anomalies of DGS1 (i.e., thymic and parathyroid hypoplasia; abnormal facial structures and cleft palate; skeletal defects; and cardiac outflow tract abnormalities), whereas Tbx1 haploinsufficiency in mutant mice (+/−) was associated only with defects of the fourth branchial pouch (i.e., cardiac outflow tract abnormalities). The basis of the phenotypic differences between DGS1 patients, who are heterozygous, and the transgenic (+/−) mice remain to be elucidated. It is plausible that Tbx1 dosage, together with the downstream genes that are regulated by Tbx1 could provide an explanation, but the roles of these putative genes in DGS1 remains to be elucidated.

Some patients may have late-onset DGS1, and these develop symptomatic hypocalcemia in childhood or during adolescence, with only subtle phenotypic abnormalities.^292,293 These late-onset DGS1 patients have similar microdeletions in the 22q11 region. It is of interest to note that the age of diagnosis in the families of the three DGS1 patients with inactivating TBX1 mutations ranged from 7 to 46 years, which is in keeping with late-onset DGS1.²⁹⁰

Hypoparathyroidism, Deafness, and Renal Anomalies Syndrome

The combined inheritance of hypoparathyroidism, deafness, and renal dysplasia (HDR) as an autosomal-dominant trait was reported in one family in 1992.²⁹⁴ Patients had asymptomatic hypocalcemia, with undetectable or inappropriately normal serum concentrations of PTH and normal brisk increases in plasma cAMP in response to the infusion of PTH. The patients also had bilateral symmetrical sensorineural deafness involving all frequencies. The renal abnormalities consisted mainly of bilateral cysts that compressed the glomeruli and tubules and led to renal impairment in some patients. Cytogenetic abnormalities were not detected, and abnormalities of the PTH gene were excluded.²⁹⁴ However, cytogenetic abnormalities involving chromosome 10p14-10pter were identified in two unrelated patients with features that were consistent with HDR. These two patients suffered from hypoparathyroidism, deafness, and growth and mental retardation. One patient also had a solitary dysplastic kidney with vesico-ureteric reflux and a uterus bicornis unicollis, and the other patient, who had a complex reciprocal insertional translocation of chromosomes 10p and 8q, had cartilaginous exostoses.²⁹⁵ Neither of these patients had immunodeficiency or heart defects, which are key features of DGS2 (see earlier), and further studies defined two non-overlapping regions; thus, the DGS2 region was located on 10p13-14 and HDR on 10p14-10pter. Deletion mapping studies in two other HDR patients further defined a critical 200-kb region that contained GATA3,²⁹⁵ which belongs to a family of zinc finger transcription factors that are involved in vertebrae embryonic development. DNA sequence analysis in other HDR patients identified mutations that resulted in a haploinsufficiency and loss of GATA3 function.^295–298 GATA3 has two zinc fingers; the C-terminal finger (ZnF2) binds DNA, and the N-terminal finger (2n-F1) stabilizes this DNA binding and interacts with other zinc finger proteins, such as the Friends of GATA (FOG).²⁹⁹ HDR-associated mutations involving GATA3 ZnF2 or the adjacent basic amino acids were found to result in a loss of DNA binding, whereas those involving ZnF1 either lead to a loss of interaction with FOG2 ZnFs or altered DNA binding affinity.^297,298,300 These findings are consistent with the proposed three-dimensional model of GATA3 ZnF1, which has separate DNA-binding and protein-binding surfaces.^297,298,301

Thus, the HDR-associated GATA3 mutations can be subdivided into two broad classes that depend upon (1) whether they disrupt ZnF1 or ZnF2 and (2) their subsequent effects on interactions with FOG2 and altered DNA binding, respectively. The majority (>75%) of these HDR-associated mutations are predicted to result in truncated forms of the GATA3 protein. Each proband and family will generally have its own unique mutation, and there appears to be no correlation with the underlying genetic defect and the phenotypic variation (e.g., presence or absence of renal dysplasia). Over 90% of patients with two or three of the major clinical features of the HDR syndrome—hypoparathyroidism, deafness, or renal abnormalities—have a GATA3 mutation.²⁹⁸ The remaining 10% of HDR of patients who do not have a GATA3 mutation of the coding region may harbor mutations in the regulatory sequences flanking the GATA3 gene, or else they may represent heterogeneity. The phenotypes of HDR patients with GATA3 mutations appear to be similar to those without GATA3 mutations.²⁹⁸

The HDR phenotype is consistent with the expression pattern of GATA3 during human and mouse embryogenesis in the developing kidney, otic vesicle, and parathyroids. However, GATA3 is also expressed in the developing central nervous system (CNS) and the hematopoietic organs in man and mice, and this suggests that GATA3 may have a more complex role. Indeed, studies of mice that are deleted for a Gata3 allele (+/−), or both Gata3 alleles (−/−) have revealed important roles for Gata3 in the development of the brain, spinal cord, peripheral auditory system, T-cells, fetal liver hematopoiesis, and urogenital system.³⁰² Gata3^+/− mice are viable, appear to be normal with a normal life span, and are fertile.³⁰² However, Gata3^+/− mice have hearing loss that is associated with cochlear abnormalities which consist of a significant progressive morphologic degeneration that starts with the outer hair cells at the apex and eventually involves all the inner hair cells, pillar cells, and nerve fibers.^303,304 These studies have shown that hearing loss in Gata3 haploinsufficiency commences in the early postnatal period and is progressive through adulthood, and that it is peripheral in origin and is predominantly due to malfunctioning of the outer hair cells of the cochlea.^303,304 Gata 3^−/− mice are embryonically lethal, and these null embryos die between 11 to 12 dpc.³⁰² Examination of these Gata3^−/− embryos revealed a variety of abnormalities that included massive internal bleeding resulting in anemia, marked growth retardation, severe deformities of the brain and spinal cord, a hypopigmented retina, gross aberrations in fetal liver hematopoiesis, a total block of T-cell differentiation, and a retarded or missing lower jaw area.^302,305 These Gata3^−/− mice had an anatomically normal sympathetic nervous system, yet the sympathetic ganglia lacked tyrosine hydroxylase and dopamine β-hydroxylase, which are key enzymes that convert tyrosine to l-dihydroxyphenylalanine (l-dopa), and dopamine to noradrenaline, respectively, in the catecholamine synthesis pathway. Thus, the Gata3^−/− mice lacked noradrenaline in the sympathetic neurons, and this was contributing to the early embryonic lethality.³⁰⁵ Feeding of catecholamine intermediates to the pregnant dams helped to partially rescue the Gata3^−/− embryos to 12.5 to 16.5 dpc. These older, pharmacologically rescued Gata3^−/− embryos showed abnormalities that could not be detected in the untreated mice.³⁰⁵ These late embryonic defects included thymic hypoplasia, a thin-walled ventricular septum, a poorly developed mandible, other developmental defects in structures derived from the cephalic neural crest cells, renal hypoplasia, a failure to form the metanephros, and an aberrant elongation of the nephric duct along the anteroposterior axis of the embryo.^305,306 The defect of the nephric duct, which consisted of an abnormal morphogenesis and guidance in the developing kidney, was characterized by the loss of Ret expression that is an essential component of the glial-derived nerve factor (GDNF) signaling pathway involved in ureteric bud formation and nephric duct guidance.³⁰⁶ Thus, Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis, as well as key regulation of nephric duct morphogenesis and guidance of the nephric duct in its caudal extension in the pro/mesonephric kidney.^305,306

It is important to note that HDR patients with GATA3 haploinsufficiency do not have immune deficiency, and this suggests that the immune abnormalities observed in some patients with 10p deletions are most likely to be caused by other genes on 10p. Similarly, the facial dysmorphism and growth and developmental delay commonly seen in patients with larger 10p deletions were absent in the HDR patients with GATA3 mutations, further indicating that these features were likely due to other genes on 10p.²⁹⁵ These studies of HDR patients clearly indicate an important role for GATA3 in parathyroid development and in the etiology of hypoparathyroidism.

Mitochondrial Disorders Associated With Hypoparathyroidism

Hypoparathyroidism has been reported to occur in three disorders associated with mitochondrial dysfunction: the Kearns-Sayre syndrome (KSS), the MELAS syndrome, and a mitochondrial trifunctional protein deficiency syndrome (MTPDS). KSS is characterized by progressive external ophthalmoplegia and pigmentary retinopathy before the age of 20 years and is often associated with heart block or cardiomyopathy. The MELAS syndrome consists of a childhood onset of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. In addition, varying degrees of proximal myopathy can be seen in both conditions. Both the KSS and MELAS syndromes have been reported to occur with insulin-dependent diabetes mellitus and hypoparathyroidism.^307,308 A point mutation in the mitochondrial gene tRNA leucine (UUR) has been reported in one patient with the MELAS syndrome who also suffered from hypoparathyroidism and diabetes mellitus.²¹⁹ Large deletions, consisting of 6741 and 6903 bp and involving more than 38% of the mitochondrial genome, have been reported in other patients who suffered from KSS, hypoparathyroidism, and sensorineural deafness.³⁰⁹ Rearrangements and duplication of mitochondrial DNA have also been reported in KSS. Mitochondrial trifunctional protein deficiency (MTPDS) is a disorder of fatty acid oxidation that is associated with peripheral neuropathy, pigmentary retinopathy, and acute fatty liver degeneration in pregnant women who carry an affected fetus. Hypoparathyroidism has been observed in one patient with trifunctional protein deficiency.³¹⁰ The role of these mitochondrial mutations in the etiology of hypoparathyroidism remains to be further elucidated.

Kenney-Caffey, Sanjad-Sakati, and Kirk-Richardson Syndromes

Hypoparathyroidism has been reported to occur in over 50% of patients with the Kenney-Caffey syndrome, which is associated with short stature, osteosclerosis and cortical thickening of the long bones, delayed closure of the anterior fontanel, basal ganglia calcification, nanophthalmos, and hyperopia.³¹¹ Parathyroid tissue could not be found in a detailed postmortem examination of one patient,³¹² and this suggests that hypoparathyroidism may be due to an embryologic defect of parathyroid development. In the Kirk-Richardson and Sanjad-Sakati syndromes, which are similar, hypoparathyroidism is associated with severe growth failure and dysmorphic features.^313,314 This has been reported in patients of Middle Eastern origin.^313,314 Consanguinity was noted in the majority of the families, indicating that this syndrome is inherited as an autosomal-recessive disorder. Homozygosity and linkage disequilibrium studies located this gene to chromosome 1q42-q43.³¹⁵ Molecular genetic investigations have identified that mutations of the tubulin-specific chaperone (TBCE) are associated with the Kenney-Caffey and Sanjad-Sakati syndromes.³¹⁶ TBCE encodes one of several chaperone proteins required for the proper folding of α-tubulin subunits and the formation of α-β tubulin heterodimers (see Fig. 5-3).³¹⁶

Additional Familial Syndromes

Single familial syndromes in which hypoparathyroidism is a component have been reported (see Table 5-1). The inheritance of the disorder in some instances has been established, and molecular genetic analysis of the PTH gene has revealed no abnormalities. An association of hypoparathyroidism, renal insufficiency, and developmental delay has been reported in one Asian family in whom autosomal-recessive inheritance of the disorder was established; an analysis of the PTH gene in this family revealed no abnormalities.³¹⁷ The occurrence of hypoparathyroidism, nerve deafness, and a steroid-resistant nephrosis leading to renal failure, which has been referred to as the Barakat syndrome,³¹⁸ has been reported in four brothers from one family, and an association of hypoparathyroidism with congenital lymphedema, nephropathy, mitral valve prolapse, and brachytelephalangy has been observed in two brothers from another family.³¹⁹ Molecular genetic studies have not been reported from these two families.

Calcium-Sensing Receptor Abnormalities

CaSR abnormalities are associated with three hypocalcemic disorders: autosomal-dominant hypocalcemic hypercalciuria (ADHH), Bartter syndrome type V (i.e., ADHH with a Bartter-like syndrome), and a form of autoimmune hypoparathyroidism due to CaSR autoantibodies (see Table 5-3).

Blomstrand’s Disease

Blomstrand’s chondrodysplasia is an autosomal-recessive human disorder characterized by early lethality, dramatically advanced bone maturation, and accelerated chondrocyte differentiation.³²⁹ Affected infants are typically born to consanguineous healthy parents (only in one instance did unrelated healthy parents have two affected offspring),^330–334 show pronounced hyperdensity of the entire skeleton (Fig. 5-9) and markedly advanced ossification, and particularly the long bones are extremely short and poorly modeled. PTH/PTHrP receptor mutations that impair its functional properties were identified as the most likely cause of Blomstrand’s disease (see Fig. 5-7). One of these defects is caused by a nucleotide exchange in exon M5 of the maternal PTH/PTHrP receptor allele, which introduces a novel splice acceptor site and thus leads to the synthesis of a receptor mutant that does not mediate, despite seemingly normal cell surface expression, the actions of PTH or PTHrP; the patient’s paternal PTH/PTHrP receptor allele is, for yet unknown reasons, only poorly expressed.³³⁵ In a second patient with Blomstrand’s disease, the product of a consanguineous marriage, a nucleotide exchange was identified that changes proline at position 132 to leucine.^336,337 The resulting PTH/PTHrP receptor mutant showed, despite reasonable cell surface expression, severely impaired binding of radiolabeled PTH and PTHrP analogs, greatly reduced agonist-stimulated cAMP accumulation, and no measurable inositol phosphate response. Additional loss-of-function mutations of the PTH/PTHrP receptor have been identified in three unrelated patients with Blomstrand’s disease. Two of these mutations led to a frameshift and a truncated protein due either to a homozygous single nucleotide deletion in exon EL2 ³³⁸ or a 27-bp insertion between exon M4 and EL2.³³⁹ The other defect was a nonsense mutation at residue 104 and thus resulted in a truncated receptor protein.³³⁹

As in Jansen’s disease, the identification of mutant PTH/PTHrP receptors provided a plausible explanation for the severe abnormalities in endochondral bone formation in patients with Blomstrand’s disease. The disease is lethal, but it is likely that besides the striking skeletal defects, affected infants show abnormalities in other organs, including secondary hyperplasia of the parathyroid glands, presumably due to hypocalcemia. In addition, analysis of fetuses with Blomstrand’s disease have revealed abnormal breast development and tooth impaction, highlighting the involvement of the PTH/PTHrP receptor in the normal development of breast and tooth.³⁴⁰

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