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-D-Galactosamine-Polypeptide N-Acetylgalactosaminyltransferase 3 in Regulating Serum Intact Fibroblast Growth Factor 23 and Matrix Extracellular Phosphoglycoprotein in Heritable Tumoral Calcinosis
Department of Medical and Molecular Genetics (H.J.G., T.E.L., S.I.D., D.L.K., K.E.W.), Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Rheumatology (C.F., B.D.), Southend General Hospital, Westcliff-on-Sea, Essex SS0 0RY, United Kingdom; Department of Endocrinology (M.J.C., M.S.D., N.C.), Saint James’s Hospital and Trinity College Dublin Medical School, Dublin 8, Ireland; and Division of Geriatric Medicine (A.J., N.S.F.), Johns Hopkins Bayview Medical Center, Baltimore, Maryland 21224
Address all correspondence and requests for reprints to: Kenneth E. White, Ph.D., 975 West Walnut Street, IB130, Indianapolis, Indiana 46202. E-mail: kenewhit{at}iupui.edu.
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Abstract |
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-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) and fibroblast growth factor-23 (FGF23) result in TC. Objective: The objective of the study was to determine the relationship between GALNT3 and FGF23 in familial TC.
Design, Setting, and Patients: We assessed the major biochemical defects and potential genes involved in patients with TC.
Intervention: Combination therapy consisted of the phosphate binder Sevelamer and the carbonic anhydrase inhibitor acetazolamide.
Results: We report a patient homozygous for a GALNT3 exon 1 deletion, which is predicted to truncate the encoded protein. This patient had high serum FGF23 concentrations when assessed with a C-terminal FGF23 ELISA but low-normal FGF23 levels when tested with an ELISA for intact FGF23 concentrations. Matrix extracellular phosphoglycoprotein has been identified as a possible regulator of phosphate homeostasis. Serum matrix extracellular phosphoglycoprotein levels, however, were normal in the family with GALNT3-TC and a kindred with TC carrying the FGF23 S71G mutation. The tumoral masses of the patient with GALNT3-TC completely resolved after combination therapy.
Conclusions: Our findings demonstrate that GALNT3 inactivation in patients with TC leads to inadequate production of biologically active FGF23 as the most likely cause of the hyperphosphatemic phenotype. Furthermore, combination therapy may be effective for reducing the tumoral burden associated with familial TC.
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Introduction |
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-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene were the first reported mutations to cause familial TC (1). GALNT3 is a UDP-N-acetyl--D-galactosamine-polypeptide N-acetylgalactosaminyltransferase that initiates mucin-like O-linked glycosylation of nascent proteins within the Golgi (2). We and others (3, 4, 5) demonstrated that the syndrome of TC was also caused by recessive mutations in the circulating phosphaturic factor fibroblast growth factor-23 (FGF23) that destabilized the intact protein and resulted in the secretion of proteolytic fragments of FGF23 (5, 6). Heritable TC is the phenotypic mirror image of the disorder autosomal dominant hypophosphatemic rickets (ADHR). ADHR is characterized by hypophosphatemia, secondary to isolated renal phosphate wasting, as well as inappropriately normal serum 1,25(OH)2D concentrations (7, 8). In contrast to the mutations that destabilize FGF23 in TC, the ADHR mutations produce an FGF23 species more resistant to intracellular proteolytic cleavage (9). In addition to hyperphosphatemia, biochemical analyses of TC patients with FGF23 mutations revealed low intact serum FGF23 protein and significantly elevated FGF23 C-terminal fragments (3). In a similar manner to FGF23-TC kindreds, TC patients with inactivating GALNT3 mutations have also been reported to have markedly elevated circulating C-terminal FGF23 levels (1). The effect of GALNT3 mutations on circulating intact FGF23 concentrations is unknown. Thus, the etiology of GALNT3-mediated TC remains unclear.
A candidate gene implicated as contributing to serum phosphate regulation is matrix extracellular phosphoglycoprotein (MEPE), which was originally isolated from a lesion that caused tumor-induced osteomalacia (10). Serum MEPE concentrations are elevated in patients with X-linked hypophosphatemic rickets (XLH) and the mouse model of XLH, the Hyp mouse (11). Furthermore, injections of recombinant MEPE have been reported to result in hypophosphatemia secondary to renal phosphate wasting in vivo (11, 12). In contrast to these results, injection of CHO cells overexpressing MEPE into nude mice did not lead to hyperphosphaturia (13). Thus, a clear role for MEPE in phosphate regulation has not been established.
The current treatment strategies for heritable TC often include surgery to remove the tumoral masses as well as a low-phosphate diet in conjunction with dietary phosphate binders and typically result in nominal success. Herein we undertook the study of a patient with TC to determine the molecular etiology of his syndrome as well as determine the circulating FGF23 and MEPE concentrations in this condition. Additionally, we examined the effects of a treatment regimen for TC consisting of an oral phosphate binder in combination with the carbonic anhydrase inhibitor acetazolamide.
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Patients and Methods |
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All patients provided written informed consent in accord with the Institutional Review Board of Indiana University. Routine serum biochemistries were assessed using standard protocols. The kindred assessed for GALNT3 and FGF23 mutations was of Cypriot descent, and the FGF23-TC kindred was Caucasian, as previously reported (3).
FGF23 serum assays
Intact FGF23 serum concentrations were determined using an ELISA according to the manufacturer’s protocol (Kainos Laboratories International, Tokyo, Japan). This assay uses monoclonal antibodies and has been shown to recognize full-length FGF23 (14). Serum FGF23 concentrations were also assessed using a C-terminal FGF23 serum assay kit (Immutopics, Inc., San Clemente, CA) (15) according to the manufacturer’s protocol. This kit is a two-site sandwich ELISA that recognizes the C-terminal portion of FGF23, thus reacting with both full-length and C-terminal fragments of FGF23.
GALNT3 and FGF23 mutational analyses
Genomic DNA was extracted from blood samples using the Qiamp DNA blood extraction kit (QIAGEN, Inc., Valencia, CA) according to the manufacturer’s protocol. Gene exons, including the intron-exon splice junctions, were PCR amplified with intronic primers (available on request) using 20 ng genomic DNA as templates. PCR conditions for all experiments were: 1 min 95 C, followed by 35 cycles of 1 min 95 C, 1 min 57 C, 1 min 72 C, and a final extension of 7 min at 72 C. Amplified exons were analyzed by DNA sequencing with the appropriate forward primers for each exon using Big Dye terminator chemistry.
GALNT3 exon 1 assessment
A PCR primer pair (forward, 5'-ATGGCTCACCTAAAGCGACT-3', reverse, 5'-TACTGCACCAAGCTTCCAGA-3') was designed to amplify the 78-bp portion of GALNT3 exon 1 containing the mutated residues. Genomic DNA samples from kindred members and controls were amplified by PCR, and the resulting products were analyzed on 3% ethidium bromide-stained agarose gels. PCR conditions were the same as those described above for mutational analyses.
Protein sequence analyses
The effects of the exon 1 mutation on the GALNT3 protein sequence were assessed using the EXPASY protein translation tool (http://ca.expasy.org/tools/).
Serum MEPE levels
Circulating intact MEPE concentrations in kindreds with GALNT3-TC and FGF23-TC were determined using a competitive ELISA, as previously reported (16).
Therapeutic protocol/evaluation
The patient was treated with Sevelamer (Renagel; Genzyme, Inc., Cambridge, MA) 1600 mg three times daily as a phosphate binding agent and the carbonic anhydrase inhibitor acetazolamide 250 mg daily to increase phosphaturia as well as dietary phosphate intake restriction.
Statistical analyses
Using SAS (version 9.1; SAS Institute, Cary, NC), the Wilcoxon rank sum test was performed to compare mean serum MEPE levels among the S71G FGF23-TC family members with the three possible genotypes: normal, carriers, and affecteds. P 
0.05 was considered significant and evidence that at least one of the genotype means was different from the mean of another genotype group.
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Results |
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The proband from the first TC family was a 26-yr-old male referred for management of a recurrent acute inflammatory polyarthritis of his shoulders, hands, and feet and tenderness and swelling of the right shoulder. A magnetic resonance imaging (MRI) indicated bilateral calcific changes over the supraspinatus, infraspinatous, and subscapularis tendons as well as cystic and degenerative changes in the humerus head and subchondral regions. Furthermore, a large ectopic calcified mass was found over the biceps brachia and infraspinatus muscles in the right shoulder. Plain radiographs showed calcification around the second metacarpophalangeal joint of the left hand, but vascular calcifications were not detectable, and the patient had normal bone mineral density. Both parents of the patient and two male siblings aged 20 and 27 yr were reported to be unaffected.
Subsequent biochemical analyses of this patient revealed hyperphosphatemia; normocalcemia; elevated 1,25(OH)2D; normal PTH concentrations; and persistent, mild hypocalciuria (Table 1
). Serum FGF23 levels were assessed using two assays: the first an ELISA that detects both full-length and C-terminal fragments of FGF23 and the second an intact ELISA that uses epitopes encompassing the 176RXXR179 furin-like protease site and recognizes only full-length FGF23 protein. The C-terminal ELISA results indicated that this patient had FGF23 concentrations greater than 20 times the normal mean, consistent with earlier reports of TC patients (1). In contrast, the intact FGF23 concentrations were found to be low-normal (Table 1).
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Genomic DNA from this patient was analyzed for FGF23 mutations as the etiology for the TC phenotype. No mutations were detected within the FGF23 exons or intronic splice sites (not shown). The lack of an FGF23 mutation then led to the examination of GALNT3 as a candidate gene because GALNT3 mutations are known to cause TC (1). Direct DNA sequence analyses of GALNT3 exons revealed a novel, homozygous, 16-bp deletion [deletion of nucleotides between nucleotides 41–58 (41–58del)] within exon 1 (Fig. 1). All other exons were negative for substitutions.
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PCR amplification of the region encompassing the 41–58del confirmed our sequencing findings. We noted the presence of a single PCR product from the patient that migrated during gel electrophoresis faster than the homologous exon 1 region from normal controls (Fig. 2). Similar amplification of the deleted region in the proband’s parents revealed that these individuals were heterozygous for the 41–58del change (Fig. 2). It was unknown, however, whether the parents were related to each other. To confirm that 41–58del was associated with the TC phenotype, amplification of 100 control alleles did not reveal this mutation. The removal of residues 41–58 was predicted to result in a frameshift that created a premature stop codon after GALNT3 residue 20 (Fig. 3). In light of the homozygous absence of residues 21–633 in the mature protein, it is likely that GALNT3 activity is lacking in this patient.
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Serum MEPE concentrations correlate with serum phosphate concentrations in normal individuals (16), and ip MEPE delivery at high concentrations in vivo may affect renal phosphate handling (12). To determine the effects of GALNT3 and FGF23 mutations on serum MEPE levels, we examined MEPE concentrations in the GALNT3-TC family described above and a kindred with an S71G FGF23-TC mutation (3). All GALNT3-TC and FGF23-TC patients as well as family members who were either homozygous normal or heterozygous for the TC-causing mutations had MEPE levels within the normal range (19–1269 ng/ml, median 476 ± 247; see Ref. 16). In this regard, the patient with GALNT3-TC and his father and mother had serum MEPE concentrations of: 246.0, 343.8, and 435.4 ng/ml, respectively. For the kindred with the FGF23 S71G mutation, the S71G homozygotes (affecteds, n = 2) had MEPE concentrations of 790 ± 90 ng/ml. The heterozygotes (carriers, n = 5) and the wild-type family members (normal, n = 6) had MEPE levels of 790 ± 170 and 680 ± 180 ng/ml, respectively. Thus, there were no statistical differences among the three genotype groups (P = 0.434). These findings indicated that MEPE may not respond to, nor be a primary contributor to, the phosphate phenotypes observed in heritable TC. MEPE levels are known to decrease with age (16). We were unable to confirm a direct correlation of MEPE values with age; however, this was most likely due to the small sample sizes of our TC kindreds (not shown).
Clinical management of TC syndrome
When initially diagnosed, as described above, the GALNT3-TC proband had radiographic evidence of a large calcified mass in the right shoulder (Fig. 4A). In an attempt to lower serum phosphate concentrations to resolve the manifestations associated with TC, this patient was treated with a combination therapy of an oral phospate binder (Sevelamer) and the carbonic anhydrase inhibitor acetazolamide. The therapeutic goal was to induce phosphaturia secondary to reducing phosphate reabsorption in the renal proximal tubule. After 8 wk of treatment, the severe shoulder calcification was completely resolved as determined by MRI (Fig. 4B). However, the reduction of this lesion was not associated with a significant change in serum phosphate or calcium concentrations. In this regard, serum phosphate values after 8, 16, and 32 wk of treatment were 6.13 mg/dl (1.98 mmol/liter), 5.97 mg/dl (1.93 mmol/liter), and 6.38 mg/dl (2.06 mmol/liter), respectively. Serum calcium concentrations at the same time points were: 9.24 mg/dl (2.31 mmol/liter), 10.36 mg/dl (2.59 mmol/liter), and 9.24 mg/dl (2.31 mmol/dl). The patient’s alkaline phosphatase was elevated at baseline, 88 U/liter (normal 20–70 U/liter), and at each of the 8-, 16-, and 32-wk follow-up measurements: 81, 82, and 105 U/liter, respectively. Reduction of the lesion was associated with other clinical improvement, including resolution of inflammatory swelling in the shoulders, hands, and feet. In this regard, the patient’s C-reactive protein acute phase marker was initially elevated at 112 mg/liter and after treatment returned to normal levels (normal range: <8 mg/liter).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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) is predicted to truncate the mature GALNT3 protein from 633 residues to 20 (Fig. 3). It is therefore highly likely that this is a loss-of-function mutation. Indeed, previously reported GALNT3 species lacking the initial 55 amino acids or the 14 C-terminal residues (amino acids 619–633) are inactive (2).
In the initial report describing the GALNT3 mutations in TC, serum FGF23 levels in patients were shown to be markedly elevated (30- to 40-fold above normal) as assessed with the C-terminal FGF23 ELISA (1). These increased FGF23 values were considered to be a physiological response to the prevailing elevated serum phosphate because intact FGF23 concentrations were not assessed. Consistent with FGF23 playing a direct role in the etiology of TC, we determined that intact FGF23 was low-normal in the face of marked hyperphosphatemia in our patient with the GALNT3 deletion (Table 1). This altered FGF23 expression pattern (i.e. elevated C-terminal and low intact FGF23 serum levels) paralleled those of patients with missense mutations in FGF23 (S71G and S129F) that resulted in degradation of this protein (3, 4, 5). These findings in our patient (who lacks an FGF23 mutation) indicated that FGF23 is misprocessed in TC, but this altered processing is due to a source outside of the FGF23 polypeptide. If the elevated FGF23 levels in GALNT3-TC were solely a response to the prevailing hyperphosphatemia and not a primary component of the pathophysiology of TC, then one would expect both the C-terminal and the intact FGF23 assays to show significant elevations in serum FGF23 concentrations. Because FGF23 is known to be O-glycosylated within the C terminus (20), GALNT3 may play a posttranslational regulatory role in the production of bioactive FGF23. Taken together, our findings indicate that the lack of GALNT3 expression directly or indirectly leads to improper FGF23 protein processing.
TC has proven a difficult disorder to treat. Common therapies include dietary restriction of phosphate, dietary phosphate binders, carbonic anhydrase inhibitors, and surgical removal of calcified masses (21, 22, 23, 24). Traditional phosphate binders, such as aluminum hydroxide, calcium carbonate, and calcium acetate, each contain elemental metals that can be absorbed into the bloodstream and incorporated into bone tissue. Of note, treatment with these binders can result in osteomalacia. Furthermore, binders containing calcium may elevate serum calcium-phosphate levels, leading to calcific deposits in soft tissue and vasculature. Surgery has not been successful in curing TC because the calcified masses often reappear. In this study, the GALNT3-TC patient was treated with combination therapy consisting of the phosphate binder Sevelamer and acetazolamide to induce phosphaturia. After treatment, the patient had resolution of his calcified masses (Fig. 4B), indicating that this regimen may prove effective for familial TC. Because serum phosphate was not significantly reduced with therapy, the mechanisms for the resolution of the calcified mass are unclear. A possible explanation for the unchanged serum phosphorus is that a melting effect of the solubilized phosphate crystals from the lesions may persistently contribute to the overall serum phosphate concentrations. Finally, whether this treatment is genotype dependent for patients carrying GALNT3 or FGF23 mutations remains to be determined.
Although MEPE has been proposed as a regulator of phosphate metabolism, in this study we did not find abnormal serum MEPE concentrations in patients with TC. If MEPE was a negative regulator of serum phosphate levels, one may expect an elevation of serum MEPE in TC. In the present study, however, despite the considerable hyperphosphatemia, all TC patients displayed MEPE levels within the normal range. Although implicated in tumor-induced osteomalacia as a potential phosphatonin, MEPE may not have a role in familial TC, and it is unknown whether MEPE is a potential target for GALNT3 glycosylation. Interestingly, average serum MEPE values in the kindred with GALNT3-TC were approximately half of those observed in the kindred with the FGF23 S71G mutation. Within the family with FGF23-TC, however, the values for MEPE in affected individuals were not statistically different from the other family members, regardless of genotype. This finding may indicate that genetic backgrounds may account for natural baseline serum MEPE differences between populations.
In summary, recessive mutations in both FGF23 and GALNT3 lead to TC. The novel GALNT3 mutation described herein is consistent with causing a truncated, nonfunctional gene product. Of significance, TC mutations in both GALNT3 and FGF23 result in a similar serum profile of low amounts of full-length, active FGF23 accompanied by elevated C-terminal fragments. These findings suggest a direct or indirect interaction between GALNT3 and FGF23 and indicate that GALNT3 activity is required for the proper processing and secretion of functional FGF23. Finally, Sevelamer, in conjunction with acetazolamide treatment, may prove to be an effective treatment for TC.
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Acknowledgments |
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Footnotes |
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H.J.G., C.F., T.E.L., S.I.D., D.L.K., M.J.C., M.S.D., N.C., A.J., N.S.F., and B.D. have nothing to declare. K.E.W. receives royalties from licensing FGF23 to Kirin Pharmaceuticals, Inc.
First Published Online July 25, 2006
Abbreviations: ADHR, Autosomal dominant hypophosphatemic rickets; 41–58del, deletion of nucleotides between nucleotides 41–58; FGF, fibroblast growth factor; GALNT3, UDP-N-acetyl--D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3; MEPE, matrix extracellular phosphoglycoprotein; MRI, magnetic resonance imaging; 1,25(OH)2D, 1,25 dihydroxyvitamin vitamin D; TC, tumoral calcinosis; XLH, X-linked hypophosphatemic rickets.
Received February 9, 2006.
Accepted July 18, 2006.
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