Physiology of Calcium, Phosphate, Magnesium and Vitamin D.

Chapter 2 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. 2nd, revised edition. Endocr Dev. Basel, Karger, 2015, vol 28, pp 7–32 (DOI: 10.1159/000380990)

Physiology of Calcium, Phosphate, Magnesium and Vitamin D Jeremy Allgrove Royal London Hospital, Honorary Consultant Paediatric Endocrinologist, Great Ormond Street Hospital, London, UK

The physiology of calcium and the other minerals involved in its metabolism is complex and intimately linked to the physiology of bone. Five principal humoral factors are involved in maintaining plasma concentrations of calcium, magnesium and phosphate and in coordinating the balance between their content in bone. The transmembrane transport of these elements is dependent on a series of complex mechanisms that are partly controlled by these hormones. The plasma concentration of calcium is initially sensed by a calciumsensing receptor, which then sets up a cascade of events that initially determines parathyroid hormone secretion and eventually results in a specific action within the target organs, mainly bone and kidney. This chapter describes the physiology of these humoral factors and relates them to the pathological processes that give rise to disorders of calcium, phosphate and magnesium metabolism as well as of bone metabolism. This chapter also details the stages in the calcium cascade, describes the effects of calcium on the various target organs, gives

details of the processes by which phosphate and magnesium are controlled and summarises the metabolism of vitamin D. The pathology of disorders of bone and calcium metabolism is described in detail in the relevant chapters. © 2015 S. Karger AG, Basel

Introduction

The metabolisms of calcium, phosphate and magnesium are intimately bound to each other; therefore, it is necessary to discuss all three together. Furthermore, this metabolism is, in many ways, different from that of most other substances by virtue of the fact that the majority of each is contained within bone, which acts as a structural material as well as a reservoir, whilst also acting as an important physiological regulator. Thus, it is required that the concentration of calcium be kept within narrow limits within plasma in order to maintain optimum neuromuscular function. Downloaded by: UCONN Storrs 198.143.38.1 - 6/17/2015 10:01:48 PM

Abstract

Calcium Physiology

A full-grown adult contains approximately 1,200 grams of calcium. In foetal and neonatal life, the total calcium content (Ca) is related to body weight (BWt), and a very close relationship exists between the two under normal circumstances. This relationship is expressed by the following formula: Ca = 0.00075*BWt1.3093

where Ca and BWt are both expressed in grams [1]. This relationship has been observed during the foetal and neonatal periods and probably largely holds true throughout the period during which bone accretion is occurring. About 99% of calcium is normally contained within bone; the remainder is present either as an intracellular cation or circulating in plasma. There are three main fractions of calcium within plasma: ionised, protein-bound and complexed (mainly to citrate or sulphate). The ionised fraction constitutes approximately 50% of the total, and most blood gas machines found within critical or intensive care units can directly measure ionised calcium. Of the remainder of the calcium in the body, most circulates bound to albumin, and plasma albumin levels affect the total concentration of calcium. Various formulae are used to ‘correct’ total calcium to allow for this, and many laboratories automatically provide a value for ‘corrected’ calcium (see Chapter 6).

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The concentration of ionised calcium is normally kept within very narrow limits (1.1–1.3 mmol/l), a level that is necessary to maintain normal neuromuscular activity. Complex mechanisms, such as altering calcium absorption in the gut, changing excretion within renal tubules and balancing the rate of deposition into or removal from bone, are involved in maintaining this concentration. If calcium levels vary significantly from this, either upwards or downwards, symptoms may develop. These issues are discussed in more detail in the relevant chapters.

Control of Plasma Calcium

Five principal humoral factors are involved in the maintenance of normal concentrations of calcium and phosphate in plasma. Plasma calcium is mainly influenced by PTH and the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D). In addition, calcitonin (CT) and parathyroid hormone-related peptide (PTHrP) play a more minor role, at least during postnatal life, but attain greater significance in a number of pathological situations. Plasma phosphate is also influenced by PTH and 1,25(OH)2D, but another factor, Fibroblast Growth Factor 23 (FGF23), also plays an important part in its metabolism. Magnesium is influenced, though to a lesser degree, by the same factors that control calcium, and it indirectly influences calcium by altering PTH secretion in response to hypocalcaemia.

Transmembrane Calcium Transport

The calcium balance is principally controlled by transport across membranes in the gastrointestinal tract and in renal tubules. The mechanisms for both are similar but exhibit differences in their emphasis, depending on which organ is involved.

Allgrove Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. 2nd, revised edition. Endocr Dev. Basel, Karger, 2015, vol 28, pp 7–32 (DOI: 10.1159/000380990)

Downloaded by: UCONN Storrs 198.143.38.1 - 6/17/2015 10:01:48 PM

Furthermore, phosphate is involved in virtually all metabolic processes, whilst magnesium is required to ensure optimum parathyroid hormone (PTH) secretion. The mechanisms required to maintain these levels are complex and dependent on a number of factors. It is the purpose of this chapter to describe these factors and to indicate how disorders of function give rise to clinical problems.

Calcium TRPV5

TRPV6 Claudin 16 Claudin 19

CB28k CB9k

Mainly GI Paracellular

Mainly renal

Na+

Ca++ PMCA1b

NCX1 Ca++

Fig. 1. Schematic representation of the mechanisms of calcium transport in the gut and renal tubules. Similar mechanisms are present in both tissues, although the importance of each differs between them. The principal mechanisms in the gut are shown on the right-hand side, and those that are more important in the renal tubules are shown on the left-hand side. Abbreviations are explained in the Appendix.

Transcellular Calcium Transport

The most important mechanism for calcium absorption in the gut is via active transport, and three steps are involved in this process [2]. There is initial absorption of calcium from the lumen that is followed by transcellular transport and lastly by extrusion of calcium across the basolateral membrane. A similar process involving related proteins is present in renal tubules (see below).

Two proteins, transient receptor potential V5 (TRPV5 (*606679)) and TRPV6 (*606680), which are members of the TRP channel protein family, are thought to play an important role in promoting active calcium transport [2, 3]. TRPV6 is the most important of these in the gut, whilst TRPV5 plays a larger role in renal tubules. These proteins inwardly rectify calcium channels whose affinity is greater for calcium than for magnesium. Once calcium reaches the intracellular compartment, cytosolic diffusion across the cell membrane is facilitated by two additional proteins, calbindin9K (*302020) in the gut and calbindin28K (*114050) in the kidney. These proteins bind calcium and transport it across the cytoplasm. At the basolat-

Calcium Physiology Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. 2nd, revised edition. Endocr Dev. Basel, Karger, 2015, vol 28, pp 7–32 (DOI: 10.1159/000380990)

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Transport occurs by both transcellular and paracellular mechanisms, which are summarised in figure 1.

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protect against the effects of vitamin D deficiency [6], presumably by increasing non-vitamin D-dependent absorption. Calcium reabsorption in renal tubules is largely passive and is influenced by a number of dietary factors, including a high sodium, protein or acid load, all of which increase calcium excretion. About 70% of the filtered load is reabsorbed passively in the proximal tubule in conjunction with sodium. A further 20% of the calcium in renal tubules is reabsorbed in the thick ascending loop of Henle by paracellular processes. The remaining 5–10% is reabsorbed in the distal tubule. Similar mechanisms to those in the gut are present, although TRPV5 is thought to be the major influence. Transcellular transport is facilitated by calbindins, particularly calbindin28k. At the basolateral surface, NCX1 is responsible for the more important mechanism, which is under hormonal influence, mainly by PTH. In the presence of hypoparathyroidism, treatment with active vitamin D analogues must be monitored carefully to prevent hypercalciuria.

Magnesium Metabolism

Magnesium is, like calcium, a divalent cation that is important for bone and calcium metabolism. Magnesium is normally present in plasma at a concentration of between 0.7 and 1.2 mmol/l, and adequate plasma magnesium is required for the normal secretion of PTH. For a more detailed description of this mechanism, see the section on the calcium-sensing receptor (CaSR). Magnesium absorption occurs in the small intestine by mechanisms that are very similar to those of calcium, although these mechanisms are not well understood [3]. They are summarised in figure 2. Two proteins, TRPM6 and TRPM7, which are related to the corresponding proteins involved in calcium absorption, facilitate transcellular magnesium transport. TRPM6 is present mainly in renal tubules and intestinal cells, whilst



eral surface, extrusion of calcium is facilitated by both an ATP-dependent Ca+-transporting ATPase (PMCA1b) (*108731) and by a Na+/Ca+ exchanger (NCX1) (*182305). In addition, calcium may be transported across the cell by passive diffusion or by extrusion of vesicles that are formed from calcium-calbindin complexes. TRPV6-mediated calcium absorption mainly occurs in the duodenum under conditions of depolarisation and during the non-fed state. There are vitamin D receptors in the duodenal cells, and both PMCA1b and TRPV6 are stimulated by 1,25(OH)2D. If these receptors are defective, as in Hereditary 1α,25(OH)2D-resistant Rickets (HVDRR) (#277440), calcium cannot be absorbed properly, and rickets results (see Chapter 8 and Case 19–38 for further details). Recently, an alternative, complementary transcellular mechanism has been proposed that occurs in the lower parts of the small intestine, particularly in the jejunum and ileum. This mechanism is dependent on another protein, the voltage-gated L-type calcium channel, Cav1.3, which operates under depolarised conditions. Cav1.3 is activated by the depolarising effects of the sodium/glucose co-transporter, SGLT1, and by other depolarising agents, such as amino acids and oligopeptides that are present in the fed state. Unlike TRPV6-mediated transport, Cav1.3-mediated transport is not saturable and is not dependent on 1,25(OH)2D but is determined by the concentration of luminal calcium [4]. It is therefore not surprising that no human conditions relating to abnormalities of calcium metabolism have so far been described in relation to mutations of TRPV6. Calcium absorption is also influenced by a number of other factors. In particular, absorption can be reduced in the presence of large quantities of calcium-binding agents such as phytate or oxalate [5]. Bisphosphonates also bind to calcium in the gut and, if used orally for therapeutic purposes, should be taken as far away from meals as possible. Alternatively, a high calcium intake helps to

Magnesium TRPM6

TRPM7

Claudin 16 Claudin 19

Paracellular

+

Na+ Pro-EGF EGFR

?

?

Mg++

į Ǆ

ǅ

Na-K

EGF

ATPase Mg++

Fig. 2. Schematic representation of the mechanisms of magnesium transport in the gut and renal tubules. Similar mechanisms are present in both tissues, although the importance of each differs between them. Abbreviations are explained in Appendix 1.

which is present on the basolateral membrane of the renal cells, where it is processed from ProEGF [7]. Following cleavage of Pro-EGF, EGF interacts with its receptor, the EGFR (*131550), which, amongst its other actions, stimulates magnesium absorption via TRPM6 on the luminal surface. It has recently been shown that mutations in the EGF gene disrupt the basolateral sorting of Pro-EGF, resulting in under-stimulation of TRPM6 and impaired magnesium reabsorption [8]. The resulting condition is known as Normocalciuric Renal Hypomagnesaemia (#611718) (see Chapter 6). TRPM6 activity is determined by the membrane potential, which is maintained by the


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TRPM7 is more widely distributed. Mutations in TRPM6 cause Hypomagnesaemia with Secondary Hypocalciuria (#602014) as a result of impaired magnesium absorption in the gut (see Chapter 6 and Case 19–15). Transcellular transport of magnesium is less well understood than that of calcium. Renal tubular reabsorption of magnesium mostly occurs along with calcium by passive reabsorption in the ascending loop of Henle, mainly via tight junctions (see below). Further along the renal tubule, active reabsorption takes place in the distal convoluted tubule, where TRPM6 is situated. TRPM6 is under the influence of Epidermal Growth Factor (EGF),

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urinary tract obstruction and the diuretic phase of acute renal failure. Chronic use of proton pump inhibitors may also cause hypomagnesaemia by inhibiting both active and passive gastrointestinal absorption of magnesium [10]; however, this phenomenon has not been described in children.

Paracellular Mechanisms of Cation Transport

Paracellular proteins are present within the tight junctions of epithelial membranes and act as barriers between cells. In some tissues, e.g. skin, these barriers are complete, whilst in others, e.g. the gastrointestinal tract and renal tubules, they are incomplete in that they prevent the transport of noxious agents, such as bacteria, whilst allowing passage of electrolytes etc., such as calcium and magnesium. Paracellular transport through these tight junctions is facilitated by a number of proteins, including, amongst others, the claudins. The most important of these, with reference to cations, are claudin 16 (*603959) (also known as paracellin 1) and claudin 19 (*610036). These two proteins coexist, mainly in the thick ascending loop of Henle, where they form heteromeric complexes. It was originally thought that these proteins acted in a specific cation transport mechanism, but it is now known that the transport occurs in response to a high sodium/chloride gradient [11]. It seems that the function of claudin 16 is to facilitate transport of sodium, whilst that of claudin 19 is to inhibit the transport of chloride, thus maintaining this high gradient. Claudin 16 is coded for by a gene on chromosome 3q28, and it mainly acts in renal tubules, where it also facilitates passive transport of magnesium and calcium. Mutations in this gene cause Hypomagnesaemia, Hypercalciuria and Nephrocalcinosis Syndrome (#248250). Nephrocalcinosis is also present because both magnesium and calcium are poorly reabsorbed (see Chapter 6).



voltage-gated potassium channel Kv1.1 encoded by the KCNA1 gene, which is located on chromosome 12p13.32 (*176260). Kv1.1 co-localises with TRPM6 and consists of four subunits that normally form homotetramers. Mutations in the gene result in both homotetrameric and heterotetrameric channels, which have a dominant-negative effect on the channels and result in one of the forms of Episodic Ataxia and/or Myokymia (#160120). However, it has recently been shown that this may also be associated with Autosomal Dominant Hypomagnesaemia [9]. Transcellular transport of magnesium is probably affected by proteins similar to the calbindins involved in calcium transport, but these mechanisms are not well understood. At the basolateral membrane, magnesium is transported partly by a mechanism that involves Na+/K+ ATPase, which consists of three subunits, α, β and γ, the latter of which is coded for by the FXYD2 (*601814) gene. Mutations in this gene result in the defective magnesium reabsorption found in the Autosomal Dominant Renal Hypomagnesaemia associated with Hypocalciuria Syndrome (#154020) (see Chapter 6). The thiazide-sensitive sodium chloride cotransporter is also involved in magnesium transport, and mutations in the coding gene, SLC12A3 (*600968), cause Gitelman’s Syndrome (#263800), in which hypermagnesuria is a feature. Raised urinary magnesium excretion is also present in some cases of Bartter’s Syndrome, which is caused by a variety of mutations that affect chloride and sodium reabsorption in the loop of Henle. In the last part of the renal tubules, the collecting ducts, both calcium and magnesium are again reabsorbed passively via tight junction proteins (see below). Renal tubular transport of magnesium can also be increased by several non-genetic causes, including the use of diuretics, gentamicin, mercury-containing laxatives or cisplatin, as well as diabetic ketoacidosis, kidney transplantation,

Phosphate Metabolism

A full-grown adult contains approximately 700 g phosphate. As with calcium, the total body content (PO4) of phosphate is closely related to BWt and is expressed by the formula: PO4 = 0.00037*BWt1.2409 [1]

Approximately 80% of phosphate is contained in bone. Of the remainder, 45% (9% of the total) is present in skeletal muscle, 54.5% is present in the viscera, and only 0.5% is present in extracellular fluid. Most phosphate is present in inorganic form but still plays a crucial part in many intracellular processes. In plasma, phosphate circulates in the form of phospholipids, phosphate esters, and free inorganic phosphate (Pi). Plasma Pi concentrations are not as tightly controlled as those of calcium and reflect the fluxes of phosphate entering and leaving the extracellular pool. In contrast to calcium, phosphate concentrations in plasma vary considerably during life, being highest during phases of rapid growth. Thus, the phosphate concentrations in premature infants are normally above 2.0 mmol/l (6.4 mg/dl), falling to 1.3–2.0 mmol/l (4.2–6.4 mg/dl) during infancy and childhood and to 0.7–1.3 mmol/l (2.2–4.3 mg/dl) in young adults.

It is not known precisely how phosphate is sensed in multicellular organisms, but more is known about how it is sensed in unicellular eukaryotes and prokaryotes. In bacteria, cellular phosphate uptake occurs via a phosphate transporter (Pst). The phosphate then activates a two component signalling system, which consists of a sensory histidine kinase (PhoR) that is coupled to a transcription factor (PhoB), which has downstream effects. This system is inhibited by phosphate and is stimulated by a lack of phosphate [13]. Sodium-phosphate co-transporters may also be involved in phosphate transport; however, whether such a system or a different system operates in higher eukaryotes is not yet known. Although phosphate concentrations do vary at different ages, there is tight control of phosphate that must involve a sophisticated sensing system. Phosphate transport across membranes is controlled by a series of sodium-dependent active transport mechanisms (Na/Pi co-transporters), of which three classes are known to exist. Type 1 is present at renal tubular brush borders but is not thought to have a major role in renal tubular reabsorption of phosphate. Type 2, which has three subtypes, 2a, 2b and 2c, is probably the most important for regulating phosphate absorption and reabsorption. Type 3 is present in many tissues but is thought to have more of a ‘gatekeeping’ role. Phosphate is readily absorbed throughout the small bowel by both passive and active mechanisms. Approximately 70% of phosphate in the body is absorbed via the type 2b Na/Pi co-transporter; the remainder is absorbed by passive absorption. This active transport is stimulated directly by 1,25(OH)2D and therefore indirectly by hypocalcaemia and PTH [14]. Since hypophosphataemia is a powerful stimulant of 25-hydroxyvitamin D-1-alphahydroxylase (1α-hydroxylase), phosphate deficiency itself stimulates increased absorption. However, the total amount of phosphate that is absorbed is dependent on the dietary phosphate


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The other component of the tight junction protein, claudin 19 (*610036), is coded for by a gene on 1p34.2. However, unlike claudin 16, claudin 19 is also present in other extra-renal tissues, particularly the cornea and retina. As a consequence, mutations in this gene not only cause hypomagnesaemia with nephrocalcinosis and progression to renal failure, which is often more rapid than that associated with claudin 16 mutations, but also eye abnormalities in more than 80% of patients. This is known as Renal Hypomagnesaemia with Ocular Involvement (#248190) [12] (see Chapter 6).

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age without affecting its intrinsic activity. As a consequence, circulating FGF23 levels remain high, resulting in the excessive renal phosphate loss in ADHR (#193100) (see Chapter 9 for further details). In contrast, mutations in the FGF23 molecule itself that render it inactive cause Hyperphosphataemic Familial Tumoral Calcinosis Type 2 (HFTC2) (#211900). Protection against inactivation results from O-glycosylation at position 178, which occurs under the influence of UDP-N-acetyl-alpha-Dgalactosamine:polypeptide N-acetylgalactosaminyl-transferase 3 (GALNT3) (*601756). GALNT3 is a 633-amino-acid protein that is coded for by a gene on chromosome 2q24-q31 [18], which has ten exons. GALNT3 itself has a single transmembrane spanning region and catalyses the O-glycosylation of serine and threonine residues on the native protein. The crucial role of GALNT3 in phosphate metabolism is demonstrated by the fact that inactivating mutations in this gene, resulting in FGF23 being cleaved more readily than normal and thus being unable to inhibit the reabsorption of phosphate by NaPi in renal tubules, results in either HFTC Type 1 (HFTC1) (#211900) [19] or Hyperostosis-Hyperphosphataemia Syndrome, which are allelic. The resulting hyperphosphataemia causes soft tissue calcinosis, similar to that of HFTC2 (see Chapter 9). The principal target organ of FGF23 is the renal tubule. When active, it acts on a receptor on the surface of the tubules. This receptor is part of the Fibroblast Growth Factor receptor family, FGFR1(IIIc) (*136350), and is coded for by a gene on chromosome 8p11.2-p11.1. The FGF receptors, of which four classes have been described, are involved in a wide variety of functions. Activating mutations cause Osteoglophonic Dysplasia (#166250), which may be associated with hypophosphataemia due to phosphate wasting [20] and is probably caused by secretion of FGF23 from the bone lesions that are part of the syndrome. Meanwhile, inactivating mutations in FGFR1 give rise to several conditions including



load and may be inhibited by phosphate-binding agents such as calcium acetate (Phosex®), calcium carbonate (Tetralac®) or sevelamer (Renagel®). These are of value in hyperphosphataemic states such as chronic renal failure, when phosphate absorption needs to be limited. The metabolism of phosphate has, until recently, been relatively poorly understood. However, in 1999, a new member of the Fibroblast Growth Factor family, FGF23, was discovered [15]. This protein was subsequently shown to be mutated in cases of Autosomal Dominant Hypophosphataemic Rickets (ADHR) (#193100) [16], and it is now known to play a key role in phosphate metabolism. FGF23 is derived from bone cells, particularly osteocytes, circulates in plasma and is subject to a variety of feedback mechanisms. As a result, it is now considered a classic hormone. The synthesis and secretion of FGF23 are modified by several factors, especially phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1) and bone morphogenic protein 1 (BMP1) (see below), and its inactivation results from cleavage of the molecule between the arginine and serine residues at positions 179/180, which is protected against by O-glycosylation of the threonine residue at position 178. When active, the principal target organ of FGF23 is the renal tubule, where it stimulates renal phosphate excretion and inhibits 1α-hydroxylase activity to reduce levels of 1,25(OH)2D. Hypophosphataemia also inhibits FGF23 secretion. These actions are summarised in figure 3. FGF23 (*605380), encoded by a gene on chromosome 12p13.3, is a 251-amino-acid protein that includes a 24-amino-acid signal sequence. FGF23 has a crucial cleavage site between residues arginine179 and serine180, where it is cleaved by a subtilisin/furin-like enzyme, rendering it inactive [17]. A second arginine residue is present at position 176, and mutations in either of these residues renders FGF23 resistant to cleav-

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Fig. 3. Schematic representation of the control of phosphate metabolism. Fibroblast Growth Factor 23 sits at the centre. Its secretion is influenced by several other factors, and it has to undergo modification before becoming active. The receptor of Fibroblast Growth Factor 23 on renal tubules enables it to promote phosphate excretion. Solid lines represent stimulatory effects, and interrupted lines represent inhibitory actions. The modes of inheritance are summarised below, and the different conditions caused by mutations in the different genes involved in the pathway are shown below. White number in black circle: Autosomal recessive inhibitory mutations. Black number in white circle: Autosomal dominant activating mutations. Black number in grey circle: Somatic mutations. White number in grey circle: X-linked dominant mutations. (1) Osteogenesis Imperfecta; (2) Autosomal Recessive Form of Hypophosphataemic Rickets 1; (3) Jeune Syndrome; (4) X-linked Dominant Hypophosphataemic Rickets; (5) Autosomal Dominant Hypophosphataemic Rickets; (6) Autosomal Recessive Form of Hypophosphataemic Rickets 2; (7) TIO; (8) McCune-Albright; (9) Hyperphosphataemic Familial Tumoral Calcinosis Type 2; (10) HHRH; 11) Hyperphosphataemic Familial Tumoral Calcinosis Type 1; (12) HHS; (13) Hyperphosphataemic Familial Tumoral Calcinosis Type 3; (14) Osteoglophonic Dysplasia; (15) KS/Hartsfield Syndrome. Abbreviations are explained in the Appendix.

the degenerative processes, such as atherosclerosis, osteoporosis and skin ageing, seen in CKD [23]. KL is not capable of acting as an FGF23 receptor on its own, but requires FGFR1. Similarly, FGFR1 is not active as an FGF23 receptor if KL is inhibited or mutated [22]. Rare patients who have inactivating mutations of KL, which cause HFTC Type 3 (HFTC3) (#211900), have been described. In contrast to HFTC1 and HFTC2, the concentrations of active circulating FGF23 in HFTC3 patients are high. This suppresses vita-


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one of the twenty-three forms of Kallmann Syndrome and a number of skeletal abnormalities [21]. FGFR1 is therefore not specific for FGF23. However, in 2006, another factor, α-Klotho (KL) (+604824), was found to act as a cofactor that confers specificity to FGFR1(IIIc) for FGF23 [22]. Klotho (named after the Greek Fate who spins the thread of life) is coded for by a gene on chromosome 13q12. Patients with Chronic Kidney Disease (CKD) have low renal expression of KL, and it has been suggested that this may accelerate

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five proteins, DMP1 (*241520), bone sialoprotein (*166490), osteopontin (*166490), dentin sialophosphoprotein (DSPP) (*125485) and matrix extracellular phosphoglycoprotein (MEPE) (*605912), that are all coded for by adjacent genes on chromosome 4p22. The SIBLING proteins, particularly DMP1 and DSPP, are secreted into the extracellular matrix and contribute to mineralisation through their acidic calcium binding domains. DMP1 is cleaved into two fragments, 37 kD and 57 kD in size, by BMP1 (see below), and these fragments have two particular features that are of importance to phosphate metabolism in common. The first has an acidic serine-aspartate-rich MEPE-associated (ASARM) peptide motif that is 23 residues in length, is the only known ligand for PHEX, and is an inhibitor of mineralisation. Furthermore, free ASARM can bind competitively with PHEX. The second fragment has an arginine-glycine-aspartate motif that binds to integrins on the cell surface. These interactions initiate downstream effects via the mitogen-activated protein kinase pathway [24] and inhibit FGF23 activity by facilitating its cleavage. Homozygous mutations in the DMP1 gene result in failure of this cleavage and leads to Autosomal Recessive Form of Hypophosphataemic Rickets 1 (#241520) that is clinically very similar to X-linked Dominant Hypophosphataemic Rickets and ADHR. The SIBLING proteins have a large number of phosphorylation sites that are phosphorylated by another enzyme, FAM20C (*611061), which regulates DMP1 activity. Homozygous mutations in the gene for this protein usually result in a rapid fatal bone sclerotic condition, Raine Syndrome (#259775), but there are occasional reports of survival into childhood [25]. DMP1 may act as a mechanostat that responds to changes in stresses within bone that are transmitted via the fluid-filled canaliculi within bone, where osteocytes lie. Other SIBLING proteins include MEPE (*605912), which also contains the ASARM and



min D 1α-hydroxylation and causes relative hypocalcaemia. As a consequence, PTH secretion is stimulated and may lead to hyperparathyroidism, which may require parathyroidectomy (see Chapter 9). Once the FGFR1-KL complex has been activated by FGF23, it increases renal tubular phosphate excretion by means of the NaPi-IIc/ SLC34A3 (*609826) exchanger at the luminal surface of the cells. Mutations in the NaPi-IIc exchanger result in Hereditary Hypophosphataemic Rickets with Hypercalciuria (HHRH) (#241530). However, unlike those conditions, which are associated with high FGF23 levels, low concentrations of active FGF23 are present; 1α-hydroxylase activity is therefore not inhibited. The resulting raised levels of 1,25(OH)2D not only stimulate calcium and phosphate absorption in the gut but also increase calcium excretion in renal tubules, resulting in the presence of hypercalciuria and renal stones. Unlike other forms of hypophosphataemic rickets, this condition should NOT be treated with active vitamin D metabolites, as it worsens the hypercalciuria. The secretion and initial processing of FGF23 is under the influence of several other factors that set up a cascade of events that eventually control FGF23 activity. The most important of these is PHEX (*300550). Several studies in the hyp-mouse, an animal model of X-linked Dominant Hypophosphataemic Rickets (#307800), have demonstrated that PHEX is somehow involved in the regulation of FGF23, despite the fact that it is not present in renal tubules. The precise mechanisms by which this occurs are not fully understood but may involve modification of the activity of the subtilisin/ furin enzyme activity that cleaves FGF23. PHEX is itself activated by DMP1 (see below). Whatever the precise mechanism, mutations in PHEX result in failure of FGF23 cleavage, which causes hyperphosphaturia and hypophosphataemia. The Small Integrin-Binding Ligand, N-linked Glycoproteins (SIBLING) proteins are a group of

tonucleotide Pyrophosphatase/Phosphodiesterase 1 deficiency leads to high FGF23 concentrations is not fully understood. Phosphate metabolism is also altered in CKD in childhood and may occasionally lead to arterial calcification and calciphylaxis. The cause of this altered phosphate metabolism is multifactorial but may respond to treatment with sodium thiosulphate [27]. Hypophosphataemia and rickets are also seen in several primary renal tubular abnormalities, such as Fanconi Syndrome (whatever the cause), in which a generalised proximal renal tubular defect, which results in bicarbonaturia, glycosuria and amino aciduria as well as a phosphate leak, is present. The most common inherited cause of Fanconi Syndrome is Cystinosis (#219800), and rickets may be the presenting feature of this condition, although it may disappear once renal failure supervenes and phosphate is retained. Hyperparathyroidism also causes a mild form of Fanconi Syndrome, and patients with parathyroid tumours may have a mild metabolic acidosis and aminoaciduria in addition to hypercalcaemia.

The Calcium Cascade

The concentration of calcium in plasma is normally maintained within very narrow limits. The initial stage of this process is binding of calcium to a specific CaSR. This then initiates a cascade of events that terminates in the action of PTH on its target organs (fig. 4).

The Calcium-Sensing Receptor Complex

Calcium is sensed in plasma via a specific CaSR (+601199), which consists of three components, CaSR itself, a G-protein-coupled second messenger (GNA11) (*139313) and an adaptor protein (AP2) (*602242) that internalises the G-proteincoupled receptor.


17


arginine-glycine-aspartate motifs. In some tumours, MEPE is produced in excess, and the ASARM motif may become free in the circulation to bind competitively with PHEX, thus inhibiting its cleavage and leading to Tumour-Induced Osteomalacia (TIO). These tumours can be very small and extremely difficult to identify, but if they can be found, tumour excision usually results in a cure. Some individuals with McCuneAlbright Polyostotic Fibrous Dysplasia (#174800), which is caused by somatic mutations in the α-subunit of the stimulatory G-protein (Gsα), have an associated excess phosphate excretion that is secondary to increased FGF23 by an, as yet, ill-understood mechanism. BMP1 is one of a group of metalloproteinases that contribute to the formation of extracellular matrix by cleaving the propeptides of several collagens to yield mature fibres. As a consequence, homozygous mutations in BMP1 have been shown to cause a recently described form of Osteogenesis Imperfecta (#614856) by downregulating matrix formation. However, BMP1 is also thought to contribute to phosphate regulation by cleaving DMP1 (and DSPP) precursors into active molecules [26] at a specific, highly conserved cleavage site. Another enzyme involved in phosphate metabolism is ectonucleotide pyrophosphatase/ phosphodiesterase 1 (ENPP1) (*173335), which catalyses the conversion of Pi to pyrophosphate and, as such, has an effect opposite that of tissue non-specific alkaline phosphatase (TNSALP) (*171760). Pyrophosphate is a natural inhibitor of mineralisation, and homozygous mutations of the gene cause Generalised Arterial Calcification of Infancy (#208000) in new-borns. Generalised Arterial Calcification of Infancy has an 85% mortality rate, but many of those who survive go on to develop a form of Autosomal Recessive Hypophosphataemic Rickets 2 (#613312) that is associated with raised FGF23 and is sometimes associated with calcification of the lateral spinal ligaments. The precise mechanism by which Ec-

Ca++ CaSR Parathyroid glands PTH PTH1R

The Calcium-Sensing Receptor

The CaSR (+601199) is a large molecule consisting of 1,078 amino acids that is coded for by a gene on chromosome 3q13-q21. It has a large extracellular calcium-binding domain consisting of approximately the first 610 residues, followed by a seven-transmembrane domain consisting of the next 250 residues and a further 210 residues making up the intracellular component. The receptor is present in many tissues, especially the parathyroid (PT) glands and renal tubules, but also in bone, cartilage and other tissues [28] such as the temporal, frontal and parietal lobes of the brain and the cerebellum and hippocampus [29]. When calcium binds to the extracellular domain of CaSR, it alters PTH secretion via both phospholipase Cb and G-protein second messengers. As a consequence, PTH secretion changes in a sigmoidal fashion in response to acute changes in plasma calcium levels (fig. 5), and there is a continuous tonic secretion of PTH, which maintains ionised calcium in the plasma at whatever level is ‘set’ by the CaSR [30]. Magnesium also binds to the CaSR and influences PTH secretion in a similar, but less potent, manner to that of calcium. However, severe magnesium deficiency inhibits PTH

18

Gsį, Ǆǅ Target organs – Kidney Bone (Gut)

secretion (see under GNA11, below, for further details [31]). Mutations within the CaSR gene result in either inactivation or activation of the receptor, which result in hyper- and hypocalcaemia, respectively. Inactivating mutations cause insensitivity to calcium, which shifts the curve of PTH secretion in response to plasma calcium to the right (fig. 5). As a consequence, PTH secretion is switched off at a higher concentration than normal, and hypercalcaemia results [28]. The receptors are also present in the renal tubule, and renal calcium excretion is thereby reduced. If these mutations are heterozygous, the resulting condition is known as Familial Benign Hypercalcaemia (FBH) or Familial Hypocalciuric Hypercalcaemia (FHH) (#145980) (see Chapter 7). If, however, the mutations are homozygous, a more serious condition, Neonatal Severe Hyperparathyroidism (#329200) may occur. CaSR inactivation can also occasionally occur when a heterozygous mutation is present and the mother is unaffected (see Chapter 7 for details), in which case, the condition is referred to as Neonatal Hyperparathyroidism, which is often self-limiting. Heterozygous mutations are also described as causing both Familial Isolated Hyperparathyroidism



Fig. 4. The calcium cascade. Plasma calcium concentrations are controlled by a series of events that begin with the effect of calcium on the calcium-sensing receptor and end with the response of the target organs.

80 70

Inactivating mutations

60 50 40

Intact PTH

30 20

Activating mutations

10 0

Ca ++ 0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

1.40

1.45

1.50

Fig. 5. Schematic representation of the relationship between plasma ionised calcium and parathyroid hormone secretion, as determined by the calcium-sensing receptor. Inactivating mutations generally shift the curve to the right, whilst activating mutations do so to the left.

Many of the mutations found in FBH are clustered around the aspartate- and glutamaterich regions of the extracellular domain of the molecule, and it has been postulated that this region contains low-affinity binding sites for calcium. Many FBH homologs have been found to have unique mutations. Furthermore, mutations have also been detected within the transmembrane domain but only rarely within the intracellular domain. Mutations within this latter domain may have a greater effect on the CaSR in the parathyroid glands than in the renal tubules, and patients in whom inactivating mutations are associated with hypercalciuria and PT gland hyperplasia, necessitating parathyroidectomy, have been described [36]. Similarly, most activating mutations that cause Autosomal Dominant Hypocalcaemia Type 1 (ADH1) are present within the extracellular calcium-binding domain. So far, nearly three hundred mutations, a


19


[32] and Tropical Chronic (Calcific) Pancreatitis (#608189) [33]; however, in the latter, some patients also have a mutation in another gene, SPINK1 (*167790), which is a pancreatic trypsin inhibitor. In contrast, activating mutations of the receptor shift the PTH secretion curve to the left (fig. 5), causing chronic hypocalcaemia and hypercalciuria, a condition known as ADHR Type 1 (ADHR1) (#193100) (see Case 19–2). One particular mutation causes constitutive activation of the receptor independent of the calcium concentration so that, rather than shifting the curve to the left, PTH secretion remains permanently switched off [34] (see Chapter 6). A rare form of idiopathic epilepsy (#612899) with activating mutations in the CaSR has also been described [29]. In addition, there is one description of an activating mutation associated with normocalcaemic hypercalciuria and renal stones [35].

The G-Protein Second Messenger

A second locus, located on chromosome 19p13, was identified by family linkage studies to cause FHH in patients who were found not to have mutations in the CaSR gene. It is now known that this locus codes for the alpha subunit of a member of the G-protein family that is coupled to the CaSR. This G-protein family has downstream effects that are mediated via phosphatidyl inositol and cAMP. Once calcium binds to the receptor, Gq11 activity is inhibited, and vice versa. Moreover, heterozygous mutations in GNA11 result in stimulatory activity, which causes ADH Type 2 (ADH2) (#615361), whilst inactivating mutations cause FHH Type 2 (FHH2) (#145981). Both of these conditions are clinically similar to their type-1 counterparts. Both calcium and magnesium have an inhibitory effect when in high concentrations, although the effect of magnesium is two- to threefold less than that of calcium. Low concentrations of calcium stimulate PTH secretion, while moderately low magnesium concentrations (0.4–0.6 mmol/l) stimulate PTH secretion to a certain extent. This secretion is sometimes accompanied by hypocalcaemia because the moderately elevated PTH induces resistance to itself [31], and it can be shown that cAMP responses to PTH infusion under these circumstances are impaired. At very low magnesium concentrations, PTH secretion is inhibited altogether, and it seems that this paradoxical inhibition is caused by constitutive activation of the Gq11 subunit by enhancing inhibitory signalling whilst having little effect on the stimulatory signalling [37, 38].

20

The Adaptor Protein

Family linkage studies of FHH also identified a third locus on chromosome 19q that is now known to be the location of a gene, AP2S1 (*602242), that codes for an adaptor protein (AP2σ1) whose function is to internalise G-protein-coupled receptors such as CaSR. AP2σ1 contains a highly conserved arginine residue at position 15 that is crucial to its function of binding to the CaSR at a dileucine motif at its C terminus. Mutations in the AP2S1 gene have been shown to cause a third form of autosomal dominant FHH (FHH3 – Oklahoma variant). Interestingly, no instances of an activating mutation of AP2S1 have been identified as a cause of putative ADH Type 3 (ADH3) [39].

The Parathyroid Glands

The PT glands, usually four in number but sometimes as many as seven, are derived embryologically from the third (lower glands) and fourth (upper glands) branchial arches. Several transcription factors are involved in their development [40]. Some, such as Hoxa3 (thyroid and thymus, chromosome 7p15-p14.2) (*142954), GATA3 (hearing sensation and kidney, chromosome 10p13–14) (*131320), TBX1 (thymus, cardiac outflow tract and the face, chromosome 22q11) (*602054) and UDF1L, are involved in the development of other structures. The latter two genes are located on the long arm of chromosome 22. Mutations within or deletion of the genes responsible for these factors result in the congenital hypoparathyroidism that is associated with other conditions such as Hypoparathyroidism, Deafness, Renal Anomalies Syndrome (#146255) (Case 19–10) and the 22q Deletion Complex, a part of DiGeorge Syndrome (#188400) (see Cases 19–3; 19–4). The homologue of Drosophila Glial Cells Missing 2 (GCM2) (*603716) is a highly conserved gene that is necessary for PT gland development and has no other known function in man. Mutations in this



few of which are polymorphisms, have been described as causing one or another of the conditions described above, and an online database has been established to keep track of them (http://www.casrdb.mcgill.ca).

gene cause Familial Isolated Hypoparathyroidism (#146200), which, in most cases, is autosomal recessive but can sometimes be autosomal dominant [41] (see Cases 19–7; 19–8; 19–9). It is also thought that the SRY-related HMG-box gene 3 (*313430), located on the X-chromosome, is involved in PT gland development and that mutations in this gene may be responsible for X-linked recessive Familial Isolated Hypoparathyroidism (%307700). Apart from these autosomal and X-linked syndromes, several mitochondrial genes are involved in PT gland development, and mutations in these genes give rise to a variety of syndromes in which hypoparathyroidism is a feature. Because the genes are mitochondrial, these syndromes are maternally inherited. For full details of these conditions, see Chapter 6 and Case 19–11. In addition to these genetic causes, destruction of the PT glands may occur as a result of surgery (e.g. following thyroidectomy), infiltration (e.g. with iron in β-thalassaemia) or antibody use. These causes may either be isolated or associated with autoantibodies to other organs, as in Polyendocrinopathy Type 1 Syndrome, also known as Autoimmune Polyendocrinopathy Syndrome (#240300) (see Chapter 6 for further details).

thesised. Mutations in the PTH gene involving the pre-pro- sequence, which result in both autosomal dominant and autosomal recessive hypoparathyroidism, have been described (#146200) (see Chapter 6). Only the first 34 N-terminal amino acids of PTH are required for full activity, and the function of the remainder of the molecule is not understood, although it has been suggested that there may be differential binding sites for the N- and Cterminal fragments in bone cells [45] and renal tubules [46]. The half-life of PTH in the circulation is 1–2 minutes [44]. The molecule is cleaved at various sites, which results in a number of fragments that can be identified in the circulation. The best modern assays of PTH measure ‘intact’ PTH, are able to measure physiological concentrations of PTH, correlate well with bioactivity and ignore the inactive fragments. This is particularly important in conditions such as chronic renal failure, where inactive fragments are cleared less rapidly than normal. The normal levels of PTH in the circulation are about 1–6 pmol/l (10–60 pg/ml) but vary depending on the assay used.

The Parathyroid Hormone Receptors

PTH acts via two receptors. The first and principal receptor is PTH1R (also called PTH/PTHrP) (*168468), which has equal affinity for both PTH and PTHrP. PTH1R consists of 593 amino acids and is encoded by a gene on the long arm of chromosome 3 [47]. It has an extracellular binding domain of 190 residues, a seven-transmembrane domain, and a cytosolic component of 134 residues. Both inactivating and activating mutations of the PTH1R have been described and result in the very rare conditions of Blomstrand Lethal Chondrodysplasia (#215045) and Jansen Disease (#156400), respectively (See Case 19–25). A second PTH2 receptor (PTH2R) is present in the central nervous system; however, PTHrP is not a ligand for it.


21


Parathyroid Hormone PTH (*168450) is a single-chain polypeptide hormone containing 84 amino acids that is encoded by a gene on chromosome 11. It is synthesised by the PT glands from prepro-PTH, which has an additional 31 N-terminal amino acids. Synthesis of PTH occurs in the ribosomes, where the initial 25-amino-acid ‘pre’ sequence acts as a signal peptide to aid transport through the rough endoplasmic reticulum [42, 43]. The ‘pre’ sequence is cleaved, and pro-PTH then travels to the Golgi apparatus, where the 6-amino-acid ‘pro’ sequence is cleaved to yield the mature hormone, which is stored in secretory vesicles that fuse with the plasma membrane prior to secretion of the hormone [44]. Very little PTH is stored within the glands, and most of the secreted hormone is newly syn-

Intracellular signalling principally occurs by coupling of the cytosolic component of the PTH1R to the G-protein second messengers Gs and Gq [48], which are heterotrimeric, consisting of α, β, and γ subunits. In the resting state, Gs and Gq are associated, and the Gsα subunit is bound to GDP (fig. 6a). Binding of the ligand to the receptor results in GDP being exchanged for GTP and dissociation of the Gsα subunit from the β,γ complex. Gsα is then free to stimulate adenylate cyclase, which results in an increase in intracellular cAMP and activation of the various actions of PTH via specific protein kinases (fig. 6b). The intrinsic GTPase activity associated with the Gsα subunit hydrolyses GTP to GDP, which causes reassociation of the components of the G-protein, and the cell reverts to its resting state. At the same time, phosphodiesterases, particularly PDE4D, inactivate cAMP to AMP, which switches off protein kinase activity (fig. 6c). This mechanism is common to several hormones, including thyroidstimulating hormone, gonadotrophins, and growth hormone-releasing hormone [48]. The Gsα subunit is encoded by a gene, GNAS1, (+139320), which is located on chromosome 20q13.3. This complex gene contains 13 exons that code for the Gsα subunit itself plus several other exons, known as A/B, XL, NESPAS (*610540) (which is an antisense transcript) and NESP55, which is only expressed in renal tubules. Alternative promoter use and splicing results in several different mRNA transcripts. In most tissues, these transcripts show biallelic expression, but those arising from the A/B, XL and NESPAS exons are paternally derived, whilst those arising from the NESP55 exon are maternally expressed. This differential expression results from methylation of these uniparental alleles that switches the activity of those alleles on or off in an epigenetic manner (fig. 7). In addition, another gene, Syntaxin (STX16) (*603666), which acts upstream of the GNAS complex, appears to influence the methylation of the A/B exon.

22

Mutations within the biallelic coding region (exons 2–13) of the gene result in resistance to the action of PTH, which clinically causes Pseudohypoparathyroidism Type Ia (#103580) (see Case 19–12) if they are associated with the maternally derived transcripts but cause Pseudopseudohypoparathyroidism (#612463) and/or Progressive Osseous Heteroplasia or Osteoma Cutis (#166350) if derived from paternal sources [49] (see Case 19–55). These patients frequently have resistance to other hormones whose actions are mediated via the Gsα second messenger mechanism and many display features of Albright’s Hereditary Osteodystrophy (AHO). Activating somatic mutations in the GNAS complex are responsible for McCune-Albright Syndrome (#174800). Alterations in the methylation patterns of the monoallelic exons, particularly A/B, cause Pseudohypoparathyroidism Type Ib (#603233) when they are on maternally derived alleles (see Case 19–13). Under these circumstances, there are no mutations found in the coding regions of the GNAS gene, and the patients do not usually have evidence of AHO. Mutations in STX16 have also been associated with some forms of Pseudohypoparathyroidism Type Ib, probably by influencing the methylation of the A/B exon (see Chapter 6). The cAMP generated by adenylate cyclase in response to hormone binding binds to protein kinase A (PKA), which is a tetramer formed of two regulatory (R) and two catalytic (C) subunits. Four different R subunits, R1α, R1β, R2α and R2β, are described, of which R1α is the most widely expressed. Three different C subunits, Cα, Cβ and Cγ, are also described, giving rise to a number of PKA variants. Under the influence of cAMP, the R subunits dissociate from the C subunits, resulting in their downstream effects. R1α is coded for by a gene, PRKAR1A (*188830), on chromosome 17q. Inactivating mutations in this gene may give rise to increased catalytic activity, which causes the Carney complex, or decreased activity, resulting in the Acrodysostosis Type 1 (ACRDYS1



Intracellular Signalling

PTH1R Adenylate cyclase

į GTPase

Ǆ

ATP

Ǔ

GDP GTP PKAC1

PKAC2

PKAR1

PKAR2

cAMP Phosphodiesterase

a

1 3

2

PTH

4

PTH1R

5

$GHQ\ODWHF\FODVH

į

GTPase

Ǆ

ATP

Ǔ

GTP GDP 8

PKAR1

PKAR2

PKAC1

PKAC2

7 cAMP 6 3KRVSKRGLHVWHUDVH

b

Phosphaturia

AMP

įK\GUR[\ODVH

PTH1R į GTPase

$GHQ\ODWHF\FODVH Ǆ

ATP

Ǔ

GTP GDP

PKAC1

PKAC2

PKAR1

PKAR2

cAMP 3KRVSKRGLHVWHUDVH

c

Phosphaturia

įK\GUR[\ODVH


AMP

23


Fig. 6. a Resting state. The α and βγ subunits are associated, and the physiological processes are quiescent. b Stimulation of the receptor by parathyroid hormone causes dissociation of the α subunit from the β,γ subunit, activating adenylate cyclase to convert ATP to cAMP, which in turn activates protein kinase to initiate its downstream effectors. c Phosphodiesterase 4D inactivates cAMP to AMP, and GTPase converts GTP to GDP; the cell then returns to its inactive state. (1) Autosomal Dominant Hyperparathyroidism; (2) Autosomal Recessive Hyperparathyroidism; (3) Jansen Metaphyseal Chondrodysplasia; (4) Blomstrand; (5) PsHP1a; (6) Acrodysostosis Type 2; (7) Carney Complex; (8) Acrodysostosis Type 1. (Black numbered circles indicate AR and white numbered circles indicate AD conditions).

STX16 M P

Nesp55 – +

Nespas + –

XL + –

A/B 1

2

3 3N 4 5 6 7

13

Allele- specific methylation bi-allelic (most tissues)

exons 2–13 exon 1

*Vį exons 2–13

A/B Paternal

exons 2–13 XL exons 2–13 Nespas

Maternal

exons 2–13 Nesp55

Fig. 7. Diagrammatic representation of the GNAS gene showing the different products that result from alternative splicing. Native Gsα is expressed biallelically. The A/B, XL and Nespas transcripts are principally expressed from the paternal allele, whilst the Nesp55 transcript is mainly expressed from the maternal allele. Since the latter is present in renal tubules and the former are only present in other tissues, mutations in exons 2–13 result in AHO but are associated with pseudohypoparathyroidism if they are derived from the maternal allele. If the paternal allele is the origin, pseudopseudohypoparathyroidism is the result. Alterations to the methylation pattern of the various alternative splicing products lacking mutations in exons 2–13 result in pseudohypoparathyroidism Type 1b if they are of maternal origin (adapted and reprinted from Bastepe et al. [67] with permission from Elsevier).

24

YS Type 2 (ACRDYS2 (ADOP4)) (#614613); however, these mutations are not usually associated with hormone resistance [50].

The Target Organs

The principal target organs of PTH are bone and kidney. In bone, PTH has two main effects. Under physiological conditions, it promotes bone formation via receptors on osteoblasts, while under cir-



(ADOHR)) (#101800) that is often associated with hormone resistance, similar to that of Pseudohypoparathyroidism, although the skeletal phenotype is more severe (see Case 19–14). The amount of cAMP that is available to bind to PKA is regulated by PDE4D (*600129), which converts cAMP to inactive AMP and is coded for by a gene on chromosome 5q. Inactivating mutations in this gene may result in increased enzyme activity, which reduces the availability of cAMP to dissociate the PKA complex and results in ACRD-

[51]. These studies showed that neonatal cord blood contained high PTH-like activity; however, N-terminal immunoreactivity was absent. The bioactivity was related to the positive gradient of calcium across the placenta, and the authors suggested that it was this that maintained the gradient. It had also been recognised for some time that some patients with malignancy developed hypercalcaemia with undetectable levels of PTH. Subsequently, a protein was purified from lung cancer cells that had similar biological properties as PTH, but was clearly different from PTH itself [52]. This protein was subsequently identified as parathyroid hormone-related peptide (PTHrP) (+168470). PTHrP is a 141-amino-acid polypeptide that is coded for by a gene on chromosome 12p12.1p11.2. It has some homology with PTH at its Nterminal end, but this homology diverges from PTH after residue 13. PTHrP cannot normally be measured in the circulation and has no significant classical hormone action in postnatal life but does have an important paracrine role in chondrocyte proliferation and maturation. PTHrP has equal activity as PTH on the PTH1R, and some of the changes seen in Jansen’s Metaphyseal Chondrodysplasia (#156400) (see Case 19– 25) are thought to be related to the overactivity of these receptors. PTHrP is not a ligand for the PTH2R, which is mainly present in brain. However, PTHrP is secreted by the lactating breast, and women with hypoparathyroidism who are breastfeeding may need to reduce their dose of vitamin D analogues. The principal pathological importance of PTHrP in postnatal life is as a cause of hypercalcaemia of malignancy (see Chapter 6).

Parathyroid Hormone-Related Peptide

Calcitonin

The presence of a PTH-like substance with similar biological activity but different immunological properties was originally suggested in 1985

Calcitonin (*114130) is a 31-amino-acid protein that is synthesised by the C cells of the thyroid gland and is encoded by a gene on chromosome


25


cumstances of hypocalcaemia, PTH stimulates bone resorption in order to retrieve calcium from the large reservoir within bone so that normocalcaemia can be restored. There are very few receptors for PTH in osteoclasts, and bone resorption occurs as a result of changes within the relationship between osteoblasts and osteoclasts. Both RANKL and osteoprotegerin are produced by osteoblasts. RANKL is coded for by a gene on chromosome 13q (TNFSF11 – *602642) and stimulates osteoclast differentiation, whilst osteoprotegerin, which is coded for on a gene on chromosome 8q (TNFRSF11B – *602634), acts as a dummy ligand for RANKL and inhibits its action. PTH alters the balance between the two in such a way as temporarily to change the balance in favour of bone resorption (see Chapter 3 for further details). In the absence of PTH for long periods, such as in unrecognised hypoparathyroidism, bone becomes undermineralised (see Case 19–4). Furthermore, homozygous mutations in TNFSF11 cause a benign form of osteoclast-poor Osteopetrosis (#259710), whilst homozygous mutations in TNFRSF11B result in Juvenile Paget Disease (#239000). In the nephron, PTH has three main actions. In the convoluted and straight parts of the proximal tubule, it stimulates the conversion of 25-hydroxyvitamin D (25OHD) to 1,25(OH)2D. Meanwhile, in the distal tubules, it promotes the reabsorption of both calcium and magnesium. It also promotes the excretion of phosphate, which allows the excess phosphate that is resorbed from bone by PTH to be excreted. There is also an effect of PTH on bicarbonate and amino acid reabsorption in the proximal tubule that results in the mild form of Fanconi Syndrome in hyperparathyroidism. This resolves when the hyperparathyroidism is reversed.

11p15.2-p15.1, which, by alternative splicing, also results in another protein, calcitonin gene-related peptide (CGRP). CT is mainly active in the thyroid gland, whilst CGRP plays more of a role in the hypothalamus. CT is secreted in response to hypercalcaemia and acts via a specific receptor that is coded for by a gene on chromosome 7q21.3 (*114131). The principal action of CT is to lower plasma calcium in a manner opposite to that of PTH. CT may also have a role in promoting skeletal mineralisation in the foetus but has a small physiological role in postnatal life. CT is sometimes used therapeutically to reduce plasma calcium in symptomatic hypercalcaemia, although bisphosphonates are now used more frequently for this purpose, but its principal value is as a marker of malignancy in Familial Medullary Thyroid Carcinoma (#155240).

or infantile forms of hypophosphatasia (#241500) (see Case 19–50), whilst heterozygous HN/HC or HN/HI cause the adult form (#146300). The intermediate childhood form (#241510) results from the HC/HC or HC/HI combination [54] (see Case 19–51). For a clinical description of these conditions, see Chapter 14. bTNAP is secreted by osteoblasts and promotes bone mineralisation. Circulating TNSAP is largely derived from liver and bone, and its levels in plasma during childhood reflect the growth rate [55] and are raised in the presence of rickets (see Chapter 8), in Juvenile Paget’s Disease (#239000) and in fibrous dysplasia (see Chapter 14). Low TNSAP levels are seen in hypophosphatasia, which results from mutations in the TNAP gene. A database that keeps track of these mutations (currently 194) has been established and can be accessed at http://www.sesep.uvsq.fr/Database. html.

Alkaline Phosphatase Vitamin D Metabolism

26

Although referred to as a vitamin, vitamin D is mainly available, not from dietary sources, but as a result of the action of sunlight on 7-dehydrocholesterol. UV light of wavelength 270–300 nm breaks the B-ring of the steroid molecule, creating a secosteroid. Further rearrangement of the molecule occurs by the action of body heat to create cholecalciferol (vitamin D3). Vitamin D is also available from plant sources as ergocalciferol (vitamin D2), which is synthesised from ergosterol and differs structurally from cholecalciferol only in the presence of an additional double bond in the side chain. Both compounds are then metabolised in a similar manner and are thought to be equipotent. Collectively, they are referred to as vitamin D or calciferol. Under normal circumstances, approximately 80% of vitamin D requirements are obtained from this action of sunlight, but synthesis is dependent on the amount of sunlight exposure, the



This enzyme is present in several tissues and exists as three main isoforms, intestinal (*171740), placental (*171810), and liver (tissue non-specific) (TNSAP) (*171760). A gene on chromosome 2q34–37 codes for the first two, and a gene on chromosome 1p36.1-p34 codes for the last [53]. Different post-translational modifications of the TNSAP enzyme result in the three tissue-specific forms found in bone, liver, and kidney that can be distinguished by their different isoelectric points and heat lability; the bone-specific form (bTNAP) being the least stable. It has been suggested that there are three codominant alleles (HN, HC and HI) of this enzyme and that the presence or absence of hypophosphatasia and its severity depends on which alleles are present. The HN allele is by far the most common and is homozygous in most individuals. The HI allele results in the most serious reduction in activity, whilst the HC allele is intermediate. Homozygous HI alleles result in the perinatal lethal

enzymes are distinguishable by their different affinities and capacities and by their intracellular localisation. The first to be cloned, a lowaffinity, high-capacity enzyme (CYP27A1) (*606530), is located in mitochondria. There are no reports of rickets resulting from mutations in this gene, but they do cause Cerebrotendinous Xanthomatosis (#213700). A second high-affinity, low-capacity enzyme (CYP2R1) (*608713), which is probably of greater physiological significance, is located within hepatic microsomes. CYP2R1 contains 501 amino acids and is coded for by a gene on chromosome 11p15.2 [60]. Rare cases are described of rickets associated with mutations in this gene (#600081) [61]. Two other enzymes, CYP3A4 (*124010) and CYP2J2 (*601258), probably also have some effect on 25-hydroxylase but are mainly involved in drug metabolism. The resulting product, 25OHD, circulates in plasma bound to the DBP in nanomolar concentrations. Assays of this compound give a measure of the vitamin D status because its level varies depending on the supply of vitamin D and shows a considerable annual variation, with a peak about 6 weeks after maximal exposure to sunlight. It is now generally agreed that vitamin D sufficiency is defined by a plasma concentration above 50 nmol/l [62]. 25OHD has some weak activity, which is not normally of clinical significance but may become significant in the presence of excess vitamin D (>300 nmol/l). Vitamin D 25 hydroxylase also catalyses the conversion of the synthetic vitamin D analogues 1α-hydroxy-cholecalciferol (alfacalcidol, One-Alpha®) and 1α-hydroxyergocalciferol (doxercalciferol, Hectorol®) to 1,25(OH)2D3 and 1,25(OH)2D2, respectively. 25OHD is metabolised to its active hormone 1,25(OH)2D by 25-hydroxyvitamin D 1α-hydroxylase, which is active only against metabolites that are already hydroxylated at position 25 [63]. A single enzyme that performs this metabolism has been identified and is located in convoluted and straight portions of the proxi-


27


strength of the UV light in that sunlight and skin colour. Cultural practices that necessitate substantial covering of the skin limit sunlight exposure. In addition, in temperate climates, there is insufficient UV light available in sunlight during winter months, even if skin exposure is possible. Melanin absorbs UV light of the appropriate wavelength and, since the melanophores that determine skin colour are situated in the skin above the keratinocytes that synthesise vitamin D, darker skinned individuals require a greater degree of sunlight exposure to achieve the same effect as light-skinned people [56]. There may be as much as a six-fold difference in this requirement to overcome this barrier, and if this is achieved, darker skinned individuals are equally capable of synthesising vitamin D. Sunscreens, which are widely used, also limit UV light availability. There is generally little vitamin D in food, although some oily fish have a relatively high content, and it is a common misconception that because a child is taking a ‘healthy diet’, they are not at risk of vitamin D deficiency. If sunlight exposure is halved, vitamin D intake must be trebled to compensate for this, and the only realistic way of achieving this is by giving adequate dietary supplementation. Vitamin D is stored in liver and adipose tissue. Obese subjects have lower circulating levels of vitamin D than non-obese subjects, possibly because they sequester more vitamin D in their fat stores [57]. Following synthesis, vitamin D is bound to a specific vitamin D-binding protein and passes to the liver. Native vitamin D has little biological activity and requires metabolism via two hydroxylation steps, first at the 25- and subsequently at the 1- position in order to become fully active [58]. All of the steps in vitamin D metabolism are catalysed by cytochrome P450 enzymes (fig. 8). The first step is catalysed by vitamin D 25 hydroxylases, and at least four different enzymes influence 25-hydroxylase activity [59]. These

7-Dehydrocholesterol

Diet

Sunlight Previtamin D Body heat

įK\GUR[\ cholecalciferol (alfacalcidol)

Cholecalciferol/Ergocalciferol (Vitamin D) Calcitroic acid

Vitamin D 25-hydroxylase 25-OH vitamin D K\GUR[\YLWDPLQ'įK\GUR[\ODVH

Vitamin D 25-hydroxylase

1,25(OH)2 vitamin D Vitamin D receptor Peripheral action

Vitamin D 24-hydroxylase GLK\GUR[\YLWDPLQ' WULK\GUR[\YLWDPLQ'

mal renal tubule; furthermore, its activity is present in osteoblasts, keratinocytes, and lymphohaematopoietic cells, where 1,25(OH)2D may have an autocrine or paracrine role. During foetal life, 1α-hydroxylase activity is found in the placenta, while in pathological states, it is present in the macrophages of sarcoid tissue and in subcutaneous fat necrosis (see Chapter 7 and Case 19–29). 1α-hydroxylase is a mitochondrial enzyme (CYP27B1) (*609506) consisting of 508 amino acids with considerable homology to other P450 enzymes and is encoded by a single gene on chromosome 12q13.1-q13.3. Mutations in this gene are responsible for the condition known variously as Pseudo-Vitamin D Deficiency Rickets, Vitamin D-Dependent Rickets Type I, Prader Rickets or 1α-Hydroxylase Deficiency (#264700). 1α-hydroxylase activity is stimulated by PTH via its cAMP/protein kinase actions. Hypocalcae-

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mia also stimulates 1α-hydroxylase activity; this is not a direct effect but is mediated via PTH. Plasma phosphate has a direct effect on 1α-hydroxylase activity; however, there is some evidence to suggest that this may be modulated by growth hormone or CT. The activity of 1α-hydroxylase is inhibited by FGF23. 1,25(OH)2D is a highly potent compound that circulates in picomolar concentrations. However, measurement of 1,25(OH)2D in plasma gives no measure of vitamin D status. 1,25(OH)2D synthesis is tightly controlled by the plasma calcium concentration. In order to enable changes in 1,25(OH)2D to occur rapidly, a second enzyme, 25OHD 24-hydroxylase (25OHD 24-OHase) (CYP24A1) (*126065), exists. This is yet another cytochrome P450 enzyme that can use both 25OHD and 1,25(OH)2D as substrates to form 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1α,24,25-trihydroxyvitamin



Fig. 8. Diagrammatic representation of vitamin D metabolism.

tion of osteoclasts via receptors on osteoblasts. In addition, there are receptors present in many tissues, such as skin, breast, prostate, colon, etc., that are not directly related to calcium homeostasis, and it has been postulated that 1,25(OH)2D may play a part in preventing cancers of these tissues [62]. Mutations in the vitamin D receptor occur throughout the molecule but particularly in either the ligand-binding (ligand-binding-negative) or DNA-binding (ligand-binding-positive) domains [66]. These mutations cause severe rickets, and many individuals, especially those with defects in DNA binding, also have alopecia. Originally referred to as Vitamin D-Dependent Rickets Type II (VDRR2), it is now more properly called HVDRR (#277440) (see Case 19–38). In another form of HVDRR, no mutations of the receptor have been identified, but it is thought to be caused by overexpression of a nuclear ribonucleoprotein that binds the hormone receptor complex to attenuate its action (%600785) (see Chapter 8 for details).

Summary and Conclusions

The mechanisms that are involved in maintaining normal calcium, magnesium and phosphate levels are complex and involve several different hormonal mechanisms that influence calcium, magnesium and phosphate in an independent but linked manner. Normal calcium and phosphate physiology demands that these mechanisms all function satisfactorily in order to maintain good bone health and demands a suitable milieu in which muscle and nerve function can be optimised. Disruptions in these mechanisms may be either environmental, principally due to vitamin D deficiency, or, in many instances, genetic. A thorough understanding of the physiology of these processes is required before a correct diagnosis can be made.


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D (1,24,25(OH)3D), respectively. The role of this enzyme is largely to divert the metabolism of 25OHD away from 1,25(OH)2D synthesis when this is not needed and to participate in the degradation of existing 1,25(OH)2D. This degradation process involves at least five steps that ultimately result in the formation of calcitroic acid, which is an inactive, water-soluble waste product. All of these steps are catalysed by CYP24A1 [59], which is inhibited by PTH and stimulated by 1,25(OH)2D and FGF23. Homozygous mutations in CYP24A1 have recently been shown to be responsible for some cases of Infantile Hypercalcaemia (#143880) [64] (see Case 19–28). 1,24,25(OH)3D has limited potency (about 10% of 1,25(OH)2D) and is probably an intermediate degradation metabolite of 1,25(OH)2D. The role, if any, of 24,25(OH)2D is uncertain. Some authors have argued that it has no role to play, whereas others have suggested that it may influence bone mineralisation. In addition, people of South Asian origin possess higher 25OHD 24-OHase activity than those of European origin [65], and this seems to contribute to their susceptibility to Vitamin D Deficiency Rickets. 1,25(OH)2D acts via a specific vitamin D receptor [66] (*601769) and is a member of the steroid-thyroid-retinoid superfamily of nuclear receptors, which, in many respects, is typical of this group. 1,25(OH)2D is located on the nuclear membrane and contains ligand binding, DNA binding, dimerisation, and transcriptional activation domains. Ligand binding by 1,25(OH)2D induces pseudodimerisation with the retinoid X receptor, which is encoded by a gene on chromosome 12 near the 1α-hydroxylase gene. The receptors are widely distributed in gut, parathyroid glands, chondrocytes, osteoblasts, and osteoclast precursors. 1,25(OH)2D plays a critical role in promoting calcium absorption in the small intestine, suppresses PTH secretion from the PT glands, influences growth plate mineralisation, and stimulates differentia-

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Jeremy Allgrove, MA, MD, FRCP, FRCPCH Department of Paediatric Endocrinology 8th Floor, North Tower, Royal London Hospital Whitechapel, London E1 1BB (UK) E-Mail [email protected]

Magnesium absorption: mechanisms and the influence of vitamin D, calcium and phosphate.

Effect of calcium phosphate and vitamin D₃ supplementation on bone remodelling and metabolism of calcium, phosphorus, magnesium and iron.

Physiology and biochemistry of vitamin D-dependent calcium binding proteins.

Phosphate metabolism and vitamin D.

Calcium, phosphorus, magnesium and vitamin D requirements of the preterm infant.

The physiology of vitamin D-far more than calcium and bone.

Vitamin D and calcium transport.

Renal control of calcium, phosphate, and magnesium homeostasis.

Interaction of alanyl-alanine with calcium, magnesium, and phosphate.

Renal clearance of phosphate and calcium in vitamin D-deficient chicks: effect of calcium loading, parathyroidectomy, and parathyroid hormone administration.

Calcium, magnesium, and phosphate abnormalities in the emergency department.

Interaction of calcium and phosphate decreases ileal magnesium solubility and apparent magnesium absorption in rats.

Effect of potassium magnesium citrate and vitamin B-6 prophylaxis for recurrent and multiple calcium oxalate and phosphate urolithiasis.

Vitamin D metabolism and calcium absorption.

The calcium and vitamin D controversy.

magnesium phosphate cement for bone substitute applications.

Magnesium deficit ? overlooked cause of low vitamin D status?

or Calcium Status?

[Classical actions of vitamin D: insights from human genetics and from mouse models on calcium and phosphate homeostasis].

Vitamin D and calcium regulation of epidermal wound healing.

[Vitamin D and calcium in the prevention of falls].

Effects of PTH and cAMP on renal handling of calcium, magnesium, and phosphate in the hamster.

Indices of intact serum parathyroid hormone and renal excretion of calcium, phosphate, and magnesium.

Effect of aspirin and indomethacin on the serum and urinary calcium, magnesium and phosphate.