Curr Osteoporos Rep DOI 10.1007/s11914-015-0254-3

RARE BONE DISEASE (CB LANGMAN AND E SHORE, SECTION EDITORS)

Hyperphosphatemic Familial Tumoral Calcinosis: Genetic Models of Deficient FGF23 Action Lisal J. Folsom & Erik A. Imel

# Springer Science+Business Media New York 2015

Abstract Hyperphosphatemic familial tumoral calcinosis (hFTC) is a rare disorder of phosphate metabolism defined by hyperphosphatemia and ectopic calcifications in various locations. To date, recessive mutations have been described in three genes involving phosphate metabolism: FGF23, GALNT3, and α-Klotho, all of which result in the phenotypic presentation of hFTC. These mutations result in either inadequate intact fibroblast growth factor-23 (FGF23) secretion (FGF23 or GALNT3) or resistance to FGF23 activity at the fibroblast growth factor receptor/α-Klotho complex (α-Klotho). The biochemical consequence of limitations in FGF23 activity includes increased renal tubular reabsorption of phosphate, hyperphosphatemia, and increased production of 1,25-dihydroxyvitamin D. The resultant ectopic calcifications can be painful and debilitating. Medical treatments are targeted toward decreasing intestinal phosphate absorption or increasing phosphate excretion; however, results have been variable and generally limited. Treatments that would increase FGF23 levels or signaling would more appropriately target the genetic etiologies of this disease and perhaps be more effective.

Keywords Fibroblast growth factor 23 . FGF23 . Hyperphosphatemia . Hyperphosphatemic familial tumoral calcinosis . Tumoral calcinosis This article is part of the Topical Collection on Rare Bone Disease L. J. Folsom : E. A. Imel (*) Department of Medicine, Division of Endocrinology, Indiana University School of Medicine, 1120 W. Michigan Street, Gatch Clinical Building Room 459, Indianapolis, IN 46202, USA e-mail: [email protected] L. J. Folsom e-mail: [email protected] L. J. Folsom : E. A. Imel Department of Pediatrics, Indiana University School of Medicine, Section of Pediatric Endocrinology, Riley Hospital for Children, 702 Barnhill Drive, Room 5960, Indianapolis, IN 46202, USA

Introduction Familial tumoral calcinosis (TC) is an autosomal recessive metabolic disorder characterized by the accumulation of calcium phosphate deposits in soft tissues. These calcifications can occur in a variety of tissues and body locations, especially in periarticular spaces, and can become quite debilitating. Two categories of familial tumoral calcinosis are differentiated by serum phosphate concentrations. Hyperphosphatemic familial TC (hFTC; MIM ID #211900) is characterized by elevated serum phosphate levels, while normophosphatemic familial TC (nFTC; MIM ID #610455), an autosomal recessive disorder, is differentiated by normal serum phosphate levels [1]. Patients with nFTC have soft tissue calcifications; however, they also have been found to have severe skin infections. In hFTC, the underlying defect in phosphate metabolism is increased renal reabsorption of phosphate resulting in hyperphosphatemia. Normal phosphate metabolism is largely regulated by fibroblast growth factor 23 (FGF23), and deficits in this system are implicated in hFTC. Since the discovery of FGF23 at the turn of the twenty-first century as a hormonal mediator in genetic disorders of hypophosphatemia, subsequent research led to the identification of multiple genetic defects causing hFTC. The purpose of this review is to summarize the pathophysiology and clinical consequences of hFTC, discuss current treatment options, and anticipate possible future interventions.

Phosphate Regulation Serum phosphate regulation in the human body is a multiorgan process maintained by the action of hormones on the intestine, kidney, and bones. Hormonal regulation is largely accomplished by the actions of vitamin D, parathyroid hormone (PTH), and FGF23. 25-Hydroxyvitamin D3 is enzymatically activated by 25-hydroxyvitamin D 1-alpha-hydroxylase

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to 1,25(OH)2 vitamin D3 [1,25(OH)2D3] in the proximal renal tubule (regulated by PTH and FGF23 as described below). Dietary phosphate sources are abundant, and isolated dietary deficiency is rare. In addition, absorption occurs throughout the small intestine and is generally more efficient than that of calcium with about 60–70 % of ingested phosphate absorbed through passive paracellular and active mechanisms. Sodium-dependent phosphate co-transporter type IIb (NPT2b) accounts for about half of total phosphate transport and 90 % of active phosphate transport [2]. 1,25(OH)2D3 acts in the intestine to upregulate transporters responsible for increasing both dietary calcium and phosphate absorption [3]. 1,25(OH)2D doubles the active transport processes but does not affect passive transport. A low-phosphate diet increases 1,25(OH)2D, but low phosphate intake itself may also upregulate NPT2b independently of 1,25(OH)2D, as confirmed in the vitamin D receptor null mouse model [3, 4]. At the kidneys, phosphate is freely filtered at the glomerulus but most is reabsorbed in the proximal tubule. PTH, an 84amino acid peptide hormone produced by the parathyroid glands, acts both on the kidney and the skeleton to regulate calcium and phosphate metabolism [5]. PTH decreases the brush-border membrane (BBM) surface expression of the sodium-dependent phosphate co-transporters type IIa and IIc (NPT2a and NPT2c, respectively) in the proximal tubule. These transporters determine the fractional tubular reabsorption of phosphate (TRP) [6]. When NPT2a and NPT2c trafficking to the BBM is upregulated, such as during dietary phosphate deprivation, TRP is increased, resulting in increased phosphate reabsorption and increased serum phosphate levels. When NPT2a and NPT2c trafficking is decreased, such as during dietary phosphate excess, TRP decreases and serum phosphate levels are thus lowered. PTH action in the bone increases expression of RANKL in osteoblasts, which stimulates osteoclast differentiation and function, thereby enhancing bone resorption and increasing release of phosphate and calcium from the bone [5, 7]. PTH also indirectly alters gastrointestinal phosphate absorption by stimulating the 25-hydroxyvitamin D 1-alpha hydroxylase and increasing 1,25(OH)2D3, which then upregulates genes involved in promotion of intestinal phosphate transport [7]. FGF23 is the gene located on chromosome 12p13.3 that codes for a protein composed of 251 amino acids, FGF23. It is a member of the FGF19 subfamily of hormonal fibroblast growth factors [8]. FGF23 is expressed primarily in osteocytes; however, this protein has also been detected in small amounts in the thalamus, thymus, heart, and small intestine. Only intact FGF23 has biologic activity at its receptor; FGF23 has a cleavage site where it can be processed by proprotein convertases into inactive N-terminal and C-terminal fragments before secretion into the circulation. Once translated, FGF23 undergoes additional modification in order to prevent this

cleavage; O-glycosylation of certain serine and threonine residues leads to the secretion of the biologically active form of FGF23 [9]. Absence of this O-glycosylation increases the sensitivity of FGF23 to cleavage enzymes. In certain genetic hypophosphatemic disorders, FGF23 expression is increased during deficiency of dentin matrix protein 1 (DMP1), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and phosphate-regulating protein with homology to endopeptidases on the X chromosome (PHEX). Research regarding the mechanism and effects of these proteins, or their absence, on FGF23 production is ongoing. Notably, PHEX overexpression does not alter normal FGF23 production or cause hyperphosphatemia [10]. FGF23 production is increased in response to higher dietary intake and serum levels of both phosphate and 1,25(OH)2D3 [11, 12]. Both FGF23 secretion and action are shown in Fig. 2a. Once secreted, FGF23 binds to the fibroblast growth factor receptors (FGFRs): 1c, 3c, and 4c [13, 14]. Although these receptors are widely expressed, they require a co-receptor αKlotho for hormonal FGF23 binding. α-Klotho is expressed primarily in the kidney, specifically in the distal tubules of the nephron, though some authors report proximal tubule expression as well, and in the parathyroid gland [1, 13, 14]. In the kidney, FGF23 signaling acts by decreasing surface expression of NPT2a and NPT2c in the BBM of the renal proximal tubules, similar to PTH, resulting in increased urinary phosphate and decreased serum phosphate [9]. FGF23 also acts opposite of PTH regarding vitamin D metabolism. FGF23 decreases the expression of 25-hydroxyvitamin D 1-alpha hydroxylase and increases expression of 25-hydroxyvitamin D-24-hydroxylase, which together lower 1,25(OH)2D3 levels [15]. Evidence suggests that FGF23 signaling at FGFR1c, 3c, or 4c may have different relative magnitude of effects between receptors in regard to renal phosphate reabsorption versus its effect on 1,25(OH)2D3 [16–18]. The normal physiologic response to dietary phosphate restriction results in decreased production of FGF23 and increased production of 1,25(OH)2D3 [19]. If FGF23 production is thus decreased with dietary phosphate restriction, the net physiologic effect should be increased reabsorption of phosphate by the kidney, and in the absence of FGF23 inhibition on 1,25(OH)2D3 production, increased 1,25(OH)2D3 levels facilitate intestinal absorption. In contrast, the normal response to dietary phosphate excess engages opposite changes in FGF23 and 1,25(OH)2D3 (increased and decreased, respectively) to those of phosphate restriction, thus providing a mechanism for normalizing phosphate balance by decreasing both intestinal absorption and renal tubular reabsorption. In situations where intact FGF23 is pathologically elevated (such as X-linked hypophosphatemia), decreased levels of 1,25(OH)2D3 thus result, which limits intestinal phosphate absorption and, together with decreased renal tubular reabsorption, causes decreased total serum phosphate concentrations as

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a consequence of FGF23 action. Conversely, when intact FGF23 is inadequately produced or has less than its normal physiologic actions, this results in the following biochemical profile: decreased urinary phosphate and elevated serum phosphate. Thus, high intact FGF23 levels can cause disorders of hypophosphatemia, while low intact FGF23 levels may result in disorders of hyperphosphatemia, including hFTC.

Clinical Presentation of hFTC The clinical presentation of hFTC is characterized by ectopic calcium-phosphate deposits in various locations. The clinical features of hFTC can range from very mild to extremely severe. Some patients have almost no symptoms at all and only display the biochemical profile of hFTC, while other patients have debilitating pain, as well as soft tissue calcifications requiring multiple surgeries and extensive medical interventions. Common locations for calcifications include large synovial joint spaces, upper and lower extremities, and buttocks, as well as a variety of other skeletal sites (Fig. 1). More rarely, calcifications have been noted in the retina, eyelid, vasculature, and brain [20–23]. In some patients, bone pain and cortical hyperostosis may also occur.

Genetic Mutations in hFTC Both human and animal genetic studies have identified a heterogeneous group of mutations responsible for the clinical and biochemical phenotype of hFTC. These mutations have thus far been identified in the genes coding for FGF23, GalNAc transferase 3 (GALNT3), and α-Klotho (αKL), and each impairs either intact FGF23 secretion or action at its receptor. Thus, hFTC is a disorder of either FGF23 deficiency or resistance. FGF23 has a cleavage recognition site and can be cleaved before secretion into inactive C-terminal and N-terminal fragments. Different assays are used to measure different portions of FGF23 from plasma. The most commonly used assays are two-site enzyme-linked immunosorbent assays (ELISA). The intact assay(s) for FGF23 have monoclonal antibodies binding to opposite sides of the 176RXXR179/S180 cleavage site within FGF23, consequently detecting only full-length FGF23. In contrast, an assay with polyclonal antibodies directed at motifs only on the C-terminal side of the cleavage site detects both Cterminal portion and full-length intact FGF23. A limitation of existing FGF23 assays in human samples is that the Cterminal measurement is not consistently higher than the intact FGF23 in all disease states (other than hFTC), as would be necessary for it to most accurately reflect Btotal^ FGF23. The combination of these assays and the pattern of elevation become important when discussing the etiology of hFTC (Table 1) [24–28].

Laboratory Findings in hFTC The biochemical profile is comprised of hyperphosphatemia, increased kidney reclamation of phosphate (demonstrated by an elevated TRP), and elevated or inappropriately normal serum concentrations of 1,25(OH)2 D 3 . The dysregulated 1,25(OH)2D3 causes calcium levels to trend in the upper normal or sometimes slightly high range, and PTH may be consequently somewhat suppressed. Although renal function is usually normal at diagnosis, in certain situations, renal function may be adversely affected by ectopic renal calcifications. Since all known genetic causes of hFTC involve impaired function of FGF23, the biochemical and physical phenotypes are similar regardless of which gene is involved. Fig. 1 Ectopic calcifications in two hyperphosphatemic familial tumoral calcinosis patients, each with GALNT3 mutations. a Calcifications involving bilateral hips, and b right knee in one patient, and c hyperostosis in the right tibia of another patient. Reprinted with permission from John Wiley & Sons, Inc. in reference [20]

FGF23 Loss of Function Mutations FGF23 knockout mouse models have shown that mice without FGF23 action have hyperphosphatemia and elevated serum 1,25(OH)2D3 levels [29]. These mice also have ectopic vascular and soft tissue calcifications and shortened lifespan as a consequence of these abnormalities. Multiple reports of mutations in the FGF23 gene in patients with hFTC have been published [25, 26]. These mutations cause an unstable FGF23 molecule by replacing conserved residues of the N-terminal segment of mature FGF23 and, as a consequence, results in decreased secretion of intact FGF23. Deficiency of intact FGF23 causes

Curr Osteoporos Rep Table 1

FGF23 levels in hFTC

The earliest mutations identified in patients with hFTC were located in the gene coding for GALNT3. GALNT3 mediates Olinked glycosylation of proteins and in FGF23 acts at three

sites, one of which is a furin-like convertase cleavage recognition sequence in FGF23 [30–32] (see Fig. 2). O-glycosylation in this region limits cleavage of FGF23 into inactive forms. Mutations impairing GALNT3 activity cause a lack of appropriate O-glycosylation of FGF23, which is then vulnerable to cleavage. The result of such mutations in GALNT3 is decreased intact FGF23 secretion which produces hyperphosphatemia as well as elevated 1,25(OH)2D3, consistent with the biochemical profile of hFTC. However, FGF23 gene expression is actually greatly increased in the mouse model of GALNT3 deficiency due to the effects of hyperphosphatemia [33]. Consequently, FGF23 production is high, but secretion of intact forms remains inefficient, which is reflected in the plasma FGF23 assay results. If using the C-terminal assay as an index of Btotal^ FGF23 (intact plus fragments), one can see that Btotal^ FGF23 is quite elevated, consistent with both increased expression and proteolytic cleavage in hFTC due to either GALNT3 or FGF23 mutations, while intact FGF23 remains low [20] (Table 1). As this mutation results in decreased levels of intact FGF23, the phenotypic and biochemical pictures of patients with this mutation are identical to those with mutations in FGF23 itself.

Fig. 2 a FGF23 and hyperphosphatemic familial tumoral calcinosis. Intact FGF23 undergoes O-glycosylation. In the absence of Oglycosylation, FGF23 is more easily cleaved before secretion into inactive fragments of FGF23. Mutations in either GALNT3 or FGF23 result in inefficient secretion of intact FGF23. Intact FGF23 binds to the αKlotho-FGF receptor complex on the nephron, leading to decreased expression of NPT2a and NPT2c transporters, thus increasing urinary

phosphate and decreasing serum phosphate. Future therapies could target increasing FGF23 gene expression or intact protein secretion or enhancing signaling by replacing αKlotho. b Targets of existing therapies for hFTC. Dietary phosphate restriction and phosphate binders act to decrease intestinal phosphate absorption, which decreases serum phosphate. Acetazolamide, nicotinamide and calcitonin increase renal phosphate excretion.

FGF23 mutation GALNT3 mutation αKlotho mutation

Intact FGF23 assay

C-terminal FGF23 assay

Low or low normal Low or low normal High

High High High

increased renal phosphate reabsorption and excessive 1,25(OH)2D3 production, resulting in increased intestinal calcium and phosphate absorption, leading to hyperphosphatemia and extraosseous calcium phosphate deposits. However, as evidence of increased expression to attempt to regulate phosphate, the FGF23 measured by the C-terminal assay is elevated but in this case mostly represents inactive fragments.

GALNT3 Loss-of-Function Mutations

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Hyperphosphatemia-hyperostosis syndrome, previously thought to be a separate entity from hFTC, is a syndrome consisting of elevated serum phosphate and radiographic findings of long bone demonstrating a cortical hyperostosis and periosteal reaction [32, 34, 35]. The biochemical phenotype and FGF23 pattern is identical to that of hFTC. Several studies have since found that patients with this syndrome have the same GALNT3 mutations as in hFTC, and it is now included in the hFTC spectrum [32, 35].

α-Klotho Missense Mutations FGF molecules in the FGF19 subfamily (FGF19, FGF21, and FGF23) have a similar structure and require tissue-specific coreceptors for bioactivity. These receptors are known as αKlotho (αKL) and β-Klotho (βKL). αKL reacts with FGF23 to regulate phosphate and 1,25(OH)2D3, while β-Klotho reacts with FGF19 and FGF21 to regulate glucose and lipid metabolism [36]. αKL is expressed primarily in the renal distal convoluted tubule, parathyroid gland, brain, and gonadal tissue. Mouse models with genetically deleted αKL have demonstrated increased expression of 25-hydroxyvitamin D 1-alpha hydroxylase and increased 1,25(OH)2D3 levels; along with increased expression of NPT2a in the proximal tubules of mice, resulting in increased renal phosphate reabsorption and hyperphosphatemia [37, 38]. These mouse models also have increased incidence of arteriosclerosis, central nervous system degeneration, and soft tissue calcifications. One human case has been reported of hFTC due to a recessive mutation in αKL [24]. This subject had intracranial and vascular calcifications as well as a biochemical profile consistent with hFTC and with normal kidney function. Of note, the subject was 13 years old at diagnosis and vascular calcifications in healthy patients in this age group are very rare. In contrast to human mutations in FGF23 and GALNT3 which result in low intact but elevated C-terminal FGF23 levels, this patient with αKL mutation was noted to have extreme elevations in both intact and C-terminal FGF23 levels. In fact, this pattern of elevations in both intact and C-terminal FGF23 was the clue leading the investigators to search for a cause of FGF23 resistance. Molecular analysis revealed decreased expression of αKL with decreased FGF23-dependent signaling, indicating that the αKL mutant protein may interfere with end-organ FGF23 activity [24]. Interestingly, in patients with chronic kidney disease, high serum FGF23 concentrations have also sometimes been associated with vascular calcifications, though this may not be causative and FGF23 may simply be a marker for conditions such as chronic kidney disease that predispose to vascular calcifications for other reasons [39, 40]. The mechanism for this is still unclear; however, as some patients with tumoral calcinosis also have vascular calcifications, the relationship is important to consider. It is more likely that in chronic kidney disease the

calcifications are not caused by high FGF23 itself, but rather by the high calcium-phosphate product, which is present in both chronic kidney disease with high intact FGF23 and in hFTC but with low intact FGF23 concentrations.

Normophosphatemic Tumoral Calcinosis In addition to hyperphosphatemic familial tumoral calcinosis, there is a form of normophosphatemic familial tumoral calcinosis (nFTC) that is differentiated from hFTC by normal bone mineral metabolism biochemistries. Patients with nFTC have similar phenotypic features to patients with hFTC, including calcinosis lesions. Additional reported features include hyperpigmented skin lesions and gingivitis. A group of eight patients from five different families with nFTC underwent a genome-wide marker screen in a study by Topaz et al., and in all eight subjects, a homozygous mutation was discovered in SAMD9, a gene that is expressed in endothelial cells and fibroblasts [41]. This mutation causes the substitution of glutamic acid for lysine at amino acid position 1495, resulting in decreased SAMD9 protein expression in cell cultures. Interestingly, individuals with this mutation in SAMD9 have higher levels of the SAMD9 messenger RNA, suggesting compensatory upregulation of the gene [41]. The function of the protein encoded for by SAMD9 is as of yet unknown; however, as patients with nFTC have evidence of inflammation, it is possible that SAMD9 may play a role in the body’s response to skin injury.

Treatment of hFTC The ectopic calcifications of hFTC can result in significant discomfort, deformity, and limitation of activity. Therefore, treatment of TC has centered primarily on targeting improvement in TC lesions, in addition to improving the biochemical profile. Both surgical and pharmacological treatments have been attempted, but as hFTC is a rare disease, no randomized clinical trials have been performed. All described treatments are published as either case reports or small case series with variable success rates, likely in part due to the heterogeneous patient population and non-standardized methods; these cases are shown in Table 2. Additionally, the targeted outcome measurements are quite variable, with some assessing primarily treatment effect on TC lesion size and symptomatic improvement, and others measuring success based on laboratory measurements such as changes in serum phosphate or urinary phosphate excretion. Surgical management has had inconsistent results, with some patients having complete resolution of calcinosis lesions after surgical removal and other patients requiring multiple repeated surgeries due to lesion recurrence [20, 22, 47, 49,

Calcitonin

Acetazolamide

1 1 1 1 1 1 5 1 1 1 1 1 1 1 4 1 1 1 1 1 2 1 1 1 1 2 1

Ichikawa [24] Lammoglia [43]a Garringer [27]a Finer [44]a Keskar [45]a Ichikawa [20]a Steinherz [46] Gregosiewicz [47] Janssen [48] Keskar [45]a Alves [49] Mikati [34]a Yamaguchi [50]a Dumitrescu [51]a Alkhooly [52] Mozaffarian [53, 54] Ichikawa [20] a Lammoglia [43]a Dumitrescu [51]a Garringer [27]a Lufkin [55] Finer [44] Mikati [34]a Weisinger [23] Yamaguchi [50]a Kallmeyer [56] Salvi [57]

Aluminum

Calcium

1

Ichikawa [20]a

2 7

n=no. of subjects

Sevelamer

Lufkin [42] Carmichael [22]

Low-phosphate diet

Phosphate binders

Reference

Treatment outcomes in reported cases of hFTC

Treatment

Table 2

Yes

Yes

No change

Yes

Yes Yes Yes No change

No change No change No change

Yes

Yes Yes No change Yes

Yes Yes No change

No change

Yes

Decreased serum Pi

Outcomes

No change

Yes

Yes

Decreased TRP or TmP

Yes

No change Yes Yes

Yes

No change

Increased urinary phosphate (24 h) or FePI

Increased/No change

Decreased

Decreased

Decreased

Initially increased, then decreased Decreased

No change

No change Decreased Decreased Resolved (decreased) No change

Decreased Decreased Increased Resolved (decreased)

Increased

Lesion size

Recurrence of lesion after surgery

Recurrence of lesion after surgery

Notes

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Multiple treatments were applied simultaneously. For most other treatments tested, some degree of dietary phosphate restriction was included, though this was not universally the case in all studied cases a

No change Yes No change Yes No change 1 1 1 Niacinamide/nicotinamide Risedronate Intravitrial ranibizumab injection

Dumitrescu [51]a Ichikawa [20]a McGrath [21]

Decreased serum Pi

n=no. of subjects Reference Treatment

Table 2 (continued)

Outcomes

Decreased TRP or TmP

Increased urinary phosphate (24 h) or FePI

Lesion size

Notes

Improved visual acuity (ocular tumoral calcinosis)

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52]. Based on available data, there does not appear to be a specific phenotype or genetic mutation that has been found to predict whether a particular patient will or will not have lesion recurrence after surgical management. Similarly, the current literature does not support a consistent, predictable response of a certain genotype to medical therapy. The goal of pharmacologic treatments is to alter the biochemical profile toward a smaller calcium-phosphate product, thus decreasing the available substrate for development of TC lesions and possibly resulting in eventual resorption of lesions. One important facet of treatment includes decreasing intestinal phosphate absorption either by limiting dietary phosphate ingestion with a very low phosphate diet or by binding dietary phosphate with dietary phosphate binding agents, such as those used primarily in the care of end-stage kidney disease patients. Alternative pathways for treatment would be to decrease renal tubular phosphate reabsorption or limit the release of phosphate from bone. Typically, multiple methods have been used simultaneously in an attempt to provide the best possible chance for symptomatic improvement. Targets for existing therapies are shown in Fig. 2b. In the developed world, it is difficult to reduce dietary phosphate intake due in part to the high prevalence of phosphate in processed foods. Dietary phosphate restriction as low as 400 mg daily has been attempted, but restriction was frequently combined with magnesium- or aluminum-based phosphate binders. There have been multiple case reports suggesting improvement in TC lesions with dietary phosphate restriction combined with phosphate binders; however, these have not routinely shown complete resolution or even improvement either in the TC lesions themselves or in symptoms including pain, redness, and swelling at the location of the calcification [47, 48, 53, 54, 55]. Although calcium-based phosphate binders have been attempted, it would seem wise to avoid calcium-based binders based on the potential to increase calcium absorption in the setting of disordered 1,25(OH) 2 D 3 metabolism which also occurs in hFTC. Sevelamer, a phosphate binding agent, becomes protonated in the intestine, then binds dietary phosphate to prevent absorption. Although results have been variable, sevelamer use has been described with some success in several hFTC case reports [27, 43–45]. The use of this medication can be limited; however, by potential gastrointestinal side effects including abdominal pain, vomiting, diarrhea, constipation, and flatulence. Another target of treatment described is the use of acetazolamide, a carbonic anhydrase inhibitor that increases urinary phosphate excretion [55]. Reports have variably described improvement in or resolution of TC lesions in patients treated with acetazolamide, though others have reported no change in lesions with treatment [27, 43–45, 50, 58, 59]. The use of acetazolamide can also be limited due to side effects noted in the general population of those treated, the most common

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of which include dyspepsia and loss of appetite. However paresthesias, polydipsia, headache, confusion, fever, rash, jaundice, and seizures have also been rarely described. Calcitonin has been trialed in hFTC as it also increases phosphate excretion independent of PTH [60]. Three case reports have been published that demonstrated increased fractional excretion of phosphate in patients with hFTC treated with calcitonin; however, lesions did not decrease in size and one lesion was reported to have progressed during treatment [56, 57, 60]. There is some suggestion based on data from patients with X-linked hypophosphatemia (XLH), the biochemical mirror of hFTC, that treatment with calcitonin might even have detrimental effects in hFTC [61]. Calcitonin increases serum 1,25(OH)2D3 levels, while hFTC also causes a tendency toward higher 1,25(OH)2D3 due to FGF23 deficiency. This increase in 1,25(OH)2D3 can increase intestinal absorption of phosphate which could worsen hyperphosphatemia in hFTC. One study using calcitonin in XLH also reported decreased levels of intact FGF23 after calcitonin injection, which would be undesirable in hFTC where intact FGF23 is already insufficient, and could also result in TC lesion progression [61]. Thus, despite calcitonin’s effect to increase phosphate excretion, it is unlikely to be an effective therapy for hFTC. Bisphosphonate therapy has been used in calcinosis associated with juvenile dermatomyositis with varying results [62]. Despite some case reports of success in juvenile dermatomyositis, in general, the effectiveness has been limited in actual practice. Although bisphosphonate treatment has been attempted in hFTC, there has not been any reported benefit described in TC lesions in patients with hFTC, so evidence to support the use of this medication class in hFTC is currently lacking [20, 63]. To date, there is no evidence suggesting that treatment outcomes vary by genotype. In fact, the outcome studies thus far have shown varying success rates even within the same family [22]. Currently, aggressive dietary phosphate restriction combined with phosphate binding using non-calcium-based binders would seem prudent in regard to medical management of hFTC. Adding acetazolamide has some potential to aid management; however, the effectiveness of known therapeutic regimens remains suboptimal. Additionally, in patients with vascular calcifications, it may be important to advise adequate control of blood pressure and lipids in an attempt to favorably affect modifiable cardiovascular risk factors. With developments in understanding of the molecular etiology of hFTC, it becomes more plausible to propose treatments geared toward increasing FGF23 in the setting of FGF23 and GALNT3 mutations or toward the modification of αKL in the setting of αKL mutations. The end objectives would be to improve patient symptoms of pain, swelling, and disfigurement, as well as the size of TC lesions. Potential targets for therapy, therefore, would be increasing FGF23

action or even producing synthetic FGF23 for systemic administration. Increased FGF23 would result in decreased renal reabsorption of phosphate with the surrogate goal of reducing hyperphosphatemia. Ichikawa et al. have demonstrated that it is possible to overcome the limitations of the GALNT3 defect in mice and generate sufficiently elevated intact FGF23 production to actually cause hypophosphatemia when crossbred to Phex-deficient mice [64]. Thus theoretically, it would be possible to overdrive FGF23 production in a hFTC patient to generate enough intact FGF23 to combat the hyperphosphatemia. Of note recently, cleaved αKL was demonstrated to stimulate bone FGF23 production in mice [65]. It might be possible that αKL could be an effective treatment to raise FGF23 production in the setting of all three hFTC genetic causes and at least partly replace the activity of the deficient αKL in αKL deficiency.

Conclusions Hyperphosphatemic familial tumoral calcinosis is a medically challenging, complex, genetically diverse disorder resulting in hyperphosphatemia, increased TMP, and soft tissue calcifications. The genetic causes are heterogeneous and involve mutations in the genes for FGF23, GALNT3, and αKL, resulting in either decreased FGF23 production in the case of FGF23 and GALNT3 mutations or FGF23 action in the case of αKL. Treatment of this syndrome remains unsatisfying due to both the lack of proven treatments and to variable effectiveness of available agents. As research regarding the genetic and molecular background of this disease is ongoing, it is possible that innovative treatments will be developed to address the underlying biochemical defects, resulting in significant improvement in patient quality of life and to further our overall understanding of this complex disease. Compliance with Ethics Guidelines Conflict of Interest LJ Folsom declares no conflicts of interest. EA Imel has received research support from Kyowa Hakko Kirin Pharma Inc. and Ultragenyx Pharmaceuticals, Inc. and served on an advisory board for Kyowa Hakko Kirin Pharma Inc. and Ultragenyx Pharmaceuticals. Human and Animal Rights and Informed Consent All studies by Erik Imel involving animal and/or human subjects were performed after approval by the appropriate institutional review boards. When required, written informed consent was obtained from all participants.

References 1. Farrow E, Imel EI, White K. Hyperphosphatemic familial tumoral calcinosis (FGF23, GALNT3, and alpha-Klotho). Best Pract Res Clin Rheumatol. 2011;25:735–47.

Curr Osteoporos Rep 2. DiMeglio LA, Imel EA. Calcium and phosphate: hormonal regulation and metabolism. In: Burr DB, Allen MR, eds. Basic and applied bone Biology. New York: Elsevier, Academic Press; 2014:261-282. 3. Sabbagh Y, Giral H, Caldas Y, Levi M, Schiavi SC. Intestinal phosphate transport. Adv in Chronic Kidney Dis. 2011;18(2):85–90. 4. Portale A, Halloran B, Morris JRR. Physiologic regulation of the serum concentration of 1,25-dihydroxyvitamin D by phosphate in normal men. J Clin Invest. 1989;83:1494–9. 5. Silva BC, Costa AG, Cusano NE, Kousteni S, Bilezikian JP. Catabolic and anabolic actions of parathyroid hormone on the skeleton. J Endocrinol Invest. 2011;34(10):801–10. 6. Blaine J, Weinman EJ, Cunningham R. The regulation of renal phosphate transport. Adv Chronic Kidney Dis. 2011;18(2):77–84. 7. Boron WF. The parathyroid glands and vitamin D. Medical Physiology: a cellular and molecular approach. Elsevier/Saunders 2003. 1094. 8. Itoh N, Omitz DM. Evolution of the Fgf and Fgfr gene families. Trends Genet. 2004;20:563–9. 9. Fukumoto S. Physiological regulation and disorders of phosphate metabolism—pivotal role of fibroblast growth factor 23. Intern Med. 2008;47(5):337–43. 10. Liu S, Guo R, Tu Q, Quarles LD. Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype. J Biol Chem. 2002;277(5):3686–97. 11. Burnett SM, Gunawardene SC, Bringhurst FR, et al. Regulation of Cterminal and intact FGF23 by dietary phosphate in men and women. J Bone Miner Res. 2006;21(8):1187–96. 12. Saito H, Maeda A, Ohtomo S, et al. Circulating FGF23 is regulated by 1alpha,25-dihydroxyvitamin D3 and phosphorus in vivo. J Biol Chem. 2005;280(4):2543–9. 13. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro OM. Regulation of fibroblast growth factor-23 signaling by Klotho. J Biol Chem. Jan 25 2006. 14. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444(7120):770–4. 15. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19:429–35. 16. Li H, Martin A, David V, Quarles LD. Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype. Am J Physiol Endocrinol Metab. 2011;300(3):E508–517. 17. Gattineni J, Twombley K, Goetz R, Mohammadi M, Baum M. Regulation of serum 1,25(OH)2 vitamin D3 levels by fibroblast growth factor 23 is mediated by FGF receptors 3 and 4. Am J Physiol Renal Physiol. 2011;301(2):F371–377. 18. Gattineni J, Alphonse P, Zhang Q, Mathews N, Bates CM, Baum M. Regulation of renal phosphate transport by FGF23 is mediated by FGFR1 and FGFR4. Am J Physiol Renal Physiol. 2014;306:F351– 358. 19. Haussler M, Hughes M, Baylink D, Littledike ET, Cork D, Pitt M. Influence of phosphate depletion on the biosynthesis and circulating level of 1α, 25-dihydroxyvitamin D. Adv Exp Med Biol. 1977;81: 233–50. 20. Ichikawa S, Baujat G, Seyahi A, Garoufali AG, Imel EA, Padgett LR, et al. Clinical variability of familial tumoral calcinosis caused by novel GALNT3 mutations. Am J Med Genet A. 2010;152A(4): 896–903. 21. McGrath E, Harney F, Kinsella F. An ocular presentation of familial tumoral calcinosis. BMJ Case Rep. 2010. 22. Carmichael KD, Bynum JA, Evans EB. Familial tumoral calcinosis: a forty-year follow-up on one family. J Bone Joint Surg Am. 2009;91(3):664–71.

23. Weisinger JR et al. Massive cerebral calcifications associated with increased renal phosphate reabsorption. Arch Intern Med. 1986;146(3):473–7. 24. Ichikawa S, Imel EA, Kreiter ML, Yu X, Mackenzie DS, Sorenson AH, et al. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis. J Clin Invest. 2007;117:2684–91. 25. Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B. An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet. 2005;14:385–90. 26. Larsson T, Yu X, Davis SI, Draman MS, Mooney SD, Cullen MJ, et al. A novel recessive mutation in fibroblast growth factor-23 causes familial tumoral calcinosis. J Clin Endocrinol Metab. 2005;90(4): 2424–7. 27. Garringer HJ, Fisher C, Larsson TE, Davis SI, Koller DL, Cullen MJ, et al. The role of mutant UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 in regulating serum intact fibroblast growth factor 23 and matrix extracellular phosphoglycoprotein in heritable tumoral calcinosis. J Clin Endocrinol Metab. 2006;91(10):4037–42. 28. Yancovitch A, Hershkovitz D, Indelman M, Galloway P, Whiteford M, Sprecher E, et al. Novel mutations in GALNT3 causing hyperphosphatemic familial tumoral calcinosis. J Bone Miner Metab. 2011;29(5):621–5. 29. Sitara D, Razzaque MS, Hesse M, Yoganathan S, Taquchi T, Erben RG, et al. Homozygous ablation of fibroblast growth factor-23 results in hyperphosphatemia and impaired skeletogenesis, and reverse hypophosphatemia in Phex-deficient mice. Matrix Biol. 2004;23(7): 421–32. 30. Topaz O, Shurman DL, Bergman R, Indelman M, Ratajczak P, Mizrachi M, Khamaysi Z, Behar D, Petronius D, Friedman V, Zelikovic I, Raimer S, Metzker A, Richard G, Sprecher E. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nature Genetics 2004;36:579–81. 31. Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H. Polypeptide GaINActransferase T3 and familial tumoral calcinosis: Secretion of FGF23 requires O-glycosylation. J Biol Chem 2006;281:18370–18377. 32. Frishberg Y, Ito N, Rinat C, Yamazaki Y, Feinstein S, Urakawa I, et al. Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J Bone Miner Res. 2007;22:235–42. 33. Ichikawa S, Sorenson A, Austin A, Mackenzie D, Fritz T, Moh A, et al. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (FGF23) concentrations and hyperphosphatemia despite increased FGF23 expression. Endocrinology. 2009;150(6):2543–50. 34. Mikati MA, Melhem RE, Najjar SS. The syndrome of hyperostosis and hyperphosphatemia. J Pediatr. 1981;99(6):900–4. 35. Ichikawa S, Guigonis V, Imel EA, Courouble M, Heissat S, Henley JD, Sorenson AH, Petit B, Lienhardt A, Econs MJ. Novel GALNT3 mutations causing hyperostosis-hyperphosphatemia syndrome result in low intact fibroblast growth factor 23 concentrations. J Clin Endocrinol Metab. 2007;92:1943–7. 36. Kurosu H, Choi M, Ogawa Y, Dickson AS, Goetz R, Eliseenkova AV, et al. Tissue-specific expression of beta-Klotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J Biol Chem. 2007;282:26687–95. 37. Tsujikawa H, Kurotaki Y, Fujimori T, Fukuda K, Nabeshima Y. Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. Mol Endocrinol. 2003;17:2393–403. 38. Segawa H, Yamanaka S, Ohno Y, Onitsuka A, Shiozawa K, Aranami F, Furutani J, Tomoe Y, Ito M, Kuwahata M, Imura A, Nabeshima Y, Miyamoto K. Correlation between hyperphosphatemia and type II

Curr Osteoporos Rep Na-Pi cotransporter activity in klotho mice. Am J Physiol Renal Physiol. 2007;292:F769–79. 39. Masai H, Joki N, Sugi K, Moroi M. A preliminary study of the potential role of FGF23 in coronary calcification in patients with suspected coronary artery disease. Atherosclerosis. 2013;226(1): 228–33. 40. Scialla JJ, Ling Lau W, Reilly MP, Isakova T, Yang H-Y, Crouthamel MH, et al. Fibroblast growth factor 23 is not associated with and does not induce arterial calcification. Kidney Int. 2013;83(6):1159–68. 41. Topaz O, Indelman M, Chefetz I, Geiger D, Metzker A, Altschuler Y, et al. A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis. Am J Hum Genet. 2006;79(4):759–64. 42. Lufkin EG, Kumar R, Heath 3rd H. Hyperphosphatemic tumoral calcinosis: effects of phosphate depletion on vitamin D metabolism, and of acute hypocalcemia on parathyroid hormone secretion and action. J Clin Endocrinol Metab. 1983;56(6):1319–22. 43. Lammoglia JJ, Mericq V. Familial tumoral calcinosis caused by a novel FGF23 mutation: response to induction of tubular renal acidosis with acetazolamide and the non-calcium phosphate binder sevelamer. Horm Res. 2009;71(3):178–84. 44. Finer G, Price HE, Shore RM, White KE, Langman CB. Hyperphosphatemic familial tumoral calcinosis: response to acetazolamide and postulated mechanisms. Am J Med Gent A. 2014;164(6):1545–9. 45. Keskar VS IE, Kulkarni M, Mane S, Jamale TE, Econs MJ, and Hase NK. The case: Ectopic calcification. Kidney International 2015; In Press. 46. Steinherz R, Chesney RW, Eisenstein B, Metzker A, DeLuca HF, Phelps M. Elevated serum calcitriol concentrations do not fall in response to hyperphosphatemia in familial tumoral calcinosis. Am J Dis Child. 1985;139(8):816–9. 47. Gregosiewicz A, Warda E. Tumoral calcinosis: successful medical treatment. A case report. J Bone Joint Surg Am. 1989;71(8):1244–9. 48. Janssen MC, de Sevaux RG. Tumoral calcinosis. J Inherit Metab Dis. 2010;33:91–2. 49. Alves C, Lima R. Hyperphosphatemic tumoral calcinosis: a 10-year follow-up. J Pediatr Endocrinol Metab. 2011;24(1–2):25–7. 50. Yamaguchi T, Sugimoto T, Imai Y, Fukase M, Fujita T, Chihara K. Successful treatment of hyperphosphatemic tumoral calcinosis with long-term acetazolamide. Bone. 1995;16:247S–50S. 51. Dumitrescu CEI, Kelly MH, Khosravi A, Hart TC, Brahim J, White KE, et al. A case of familial tumoral calcinosis/hyperostosishyperphosphatemia syndrome due to a compound heterozygous mutation in GALNT3 demonstrating new phenotypic features. Osteoporos Int. 2009;20(7):1273–8.

52. Alkhooly AZ. Medical treatment for tumoral calcinosis with eight years of follow-up: a report of four cases. J Orthop Surg (Hong Kong). 2009;17(3):379–82. 53. Mozaffarian G, Lafferty FW, Pearson OH. Treatment of tumoral calcinosis with phosphorus deprivation. Ann Intern Med. 1972;77:741– 5. 54. Mozaffarian G, Nakhjavani MK, Hedayati MH, Shamekh S. Phosphorus deprivation treatment of tumoral calcinosis. Ann Intern Med. 1977;86:120. 55. Lufkin EG, Wilson DM, Smith LH, Bill NJ, DeLuca HF, Dousa TP, et al. Phosphorus excretion in tumoral calcinosis: response to parathyroid hormone and acetazolamide. J Clin Endocrinol Metab. 1980;50:648–53. 56. Kallmeyer JC, Seimon LP, MacSearraigh ET. The effect of thyrocalcitonin therapy and phosphate deprivation on tumoral calcinosis. S Afr Med J. 1978;54:963–6. 57. Salvi A, Cerudelli B, Cimino A, Zuccato F, Giustina G. Phosphaturic action of calcitonin in pseudotumoral calcinosis. Horm Metab Res. 1983;15:260. 58. Knox FG, Haas JA, Lechene CP. Effect of parathyroid hormone on phosphate reabsorption in the presence of acetazolamide. Kidney Int. 1976;10:216–20. 59. Sinha TK, Allen DO, Queener SF, Bell NH, Larson S, McClintock R. Effects of acetazolamide on the renal excretion of phosphate in hypoparathyroidism and pseudohypoparathyroidism. J Lab Clin Med. 1977;89:1188–97. 60. Candrina R, Cerudelli B, Braga V, Salvi A. Effects of the acute subcutaneous administration of synthetic salmon calcitonin in tumoral calcinosis. J Endocrinol Invest. 1989;12:55–7. 61. Liu ES, Carpenter TO, Gundberg CM, Simpson CA, Insogna KL. Calcitonin administration in X-linked hypophosphatemia. N Engl J Med. 2011;364:1678–80. 62. Marco Puche A, Calvo Penades I, Lopez MB. Effectiveness of the treatment with intravenous pamidronate in calcinosis in juvenile dermatomyositis. Clin Exp Rheumatol. 2010;28:135–40. 63. Leicht E, Tkocz HJ, Seeliger H, Lauffenburger T, Haas HG. Tumoral calcinosis. Observations during six years. Horm Metab Res. 1980;12: 269–73. 64. Ichikawa S, Austin AM, Gray AK, Econs MJ, Ichikawa S, Austin AM, et al. A Phex mutation in a murine model of X-linked hypophosphatemia alters phosphate responsiveness of bone cells. J Bone Miner Res. 2012;27(2):453–60. 65. Smith RC, O’Bryan LM, Farrow EG, Summers LJ, Clinkenbeard EL, Roberts JL, et al. Circulating αKlotho influences phosphate handling by controlling FGF23 production. J Clin Invest. 2012;122(12):4710– 5.