I norganic phosphorus (Pi) plays an important role in a number of biological processes and is a major component of bone mineral1. During skeletal growth and bone modeling, calcium and phosphate are required for the formation of hydroxyapatite and, in their absence, mineralization of bone is impaired, with consequent osteomalacia and rickets2. Among the calciotropic hormones, parathyroid hormone and 1,25-dihydroxyvitamin D (1,25[OH]2D3) have been well-recognized modulators of the circulating Pi concentrations2.
The phosphatonins are a new class of hormones with a role in the regulation of Pi transport and homeostasis2,3. The term phosphatonin was introduced to describe the factor(s) responsible for the inhibition of renal Pi reabsorption and for the modulation of 25-hydroxyvitamin D 1a-hydroxylase levels4. Fibroblast growth factor-23 (FGF-23) protein, a well-recognized phosphatonin, belongs to the family of the FGF ligands. It inhibits Pi reabsorption from the renal proximal tubule and indirectly decreases Pi intestinal absorption by impairing renal 1,25(OH)2D3 synthesis5. The physiological role of FGF-23 in modulating plasma Pi concentrations and renal 1a-hydroxylase activities has been elegantly described in a study that generated Fgf23-null mice, whose growth rate was reduced, with elevated serum phosphorus and 1,25(OH)2D3 levels6. Conversely, overexpression of the FGF23 gene in transgenic mice is associated with reduced serum Pi concentrations, increased renal Pi excretion, and low expression of the renal sodium-phosphate cotransporter (NaPi-IIa)7. Indeed, activating mutations of the FGF23 gene, located on chromosome 12p13.3, as reported by Econs et al.8, were initially postulated to be responsible for autosomal hypophosphatemic rickets9, a disorder characterized by hypophosphatemia, decreased or inappropriately normal serum 1,25(OH)2D3 levels, and rickets or osteomalacia.
The mirror image phenotype of autosomal hypophosphatemic rickets is tumoral calcinosis, characterized by hyperphosphatemia due to enhanced renal tubular phosphate reabsorption and by ectopic calcifications, especially around large joints10,11. The occurrence of the disease is often associated with dental abnormalities and inappropriately normal or elevated serum levels of 1,25(OH)2D3. Other potentially related problems include anemia, low-grade fever, regional lymphadenopathy, splenomegaly, amyloidosis, and recurrent episodes of skeletal inflammation10. Recently, homozygous mutations on the UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-3 (GALNT3) gene have been recognized to be the cause of recessive forms of tumoral calcinosis12. However, the molecular mechanisms underlying the etiology of tumoral calcinosis due to GALNT3 gene mutations are not well understood. As the tumoral calcinosis phenotype is similar to that described in the Fgf23 knockout mouse6, it was not surprising that recessive mutations in the FGF23 gene were eventually described in thirteen subjects affected by this disease13,14. These findings are suggestive of a direct substrate-enzyme relationship between FGF-23 and GALNT314.
In the present study, we describe a new FGF23 gene mutation in two unrelated patients affected by tumoral calcinosis.
Patients and Controls
Case 1
A sixty-five-year-old white woman was seen for ectopic calcifications. She was 160 cm tall and weighed 64 kg. The physical examination was not noteworthy except for ectopic calcifications. A rock-hard enlargement was present on the left shoulder with consequent reduced adduction of the left arm. The left gluteal area was similarly enlarged, with flexion and internal and external rotation of the hip substantially impaired because of periarticular calcifications.
When she was twenty-five years old, the pelvic and humeral ectopic calcifications had been surgically removed. A new left pelvic calcification was removed when she was forty-five years old. Two decades later, a new 10-cm calcification that had appeared in the left shoulder was surgically removed.
The patient's parents were consanguineous, and the mother's death was caused by complications of severe arterial calcifications. The fifty-one-year-old daughter of the patient had no ectopic calcifications, with normal biochemical values, including serum phosphorus levels. The pedigree of the family is shown in Figure 1.
Case 2
A fifty-five-year-old white man was seen for ectopic calcifications. He was 175 cm tall and weighed 72 kg. The physical examination was not noteworthy except for ectopic calcifications. A mass located in the right buttock and characterized on magnetic resonance imaging by solid and fluid areas without bone infiltration recurred four years after surgical removal of a 15-cm lesion. Another ectopic calcification of the left shoulder that had occurred when he was twenty years old had been surgically removed. The parents of the patient were not consanguineous.
Control Group
A group of 100 (200 chromosomes) unrelated white individuals who had no known history of tumoral calcinosis were randomly selected at the outpatient clinic of the Metabolic Bone Disease Unit of our university and were evaluated for FGF23 gene mutations. There were fifty-eight women and forty-two men with a mean age (and standard deviation) of 61 ± 18 years. Subjects taking calcium or other supplements were excluded from the recruitment. The patients and control subjects signed an informed consent for genetic testing, in accordance with regulations of the institutional review board of our university.
Serum and Urine Biochemistry
Routine serum biochemistry was assessed with use of standard procedures. Serum concentrations of intact and C-terminal fragment of the FGF-23 were measured with use of an enzyme-linked immunoabsorbent assay (ELISA) (Immunotopics, San Clemente, California). Serum bone-specific alkaline phosphatase activity and urinary pyridinoline were also measured. Renal Pi handling was assessed by calculating the ratio of phosphate transport maximum to glomerular filtration rate (TmP/GFR) with use of serum samples obtained after an overnight fast and urine samples from twenty-four hours of urinary collection (Table I).
Dietary Intake
A detailed medical history obtained from the two patients and from the daughter of one (Case 1) included the evaluation of the dietary phosphate and calcium intake assessed by a validated food questionnaire (Table I). The patients did not consume supplemental sources of either calcium or phosphorus.
Radiographic Studies
Figure 2 shows a radiographic image of the left shoulder calcification of the patient in Case 1 before and after surgical removal.
Areal bone mineral density (g/cm2) was determined for the lumbar spine (L1-L4) and the femoral neck by dual x-ray absorptiometry with use of a Hologic 4500 machine (Hologic, Waltham, Massachusetts), with a short-term in vivo coefficient of variation of 0.9%.
In both patients, arterial and venous Doppler ultrasonography of the lower extremities was carried out.
Histopathology
Histopathological findings of the ectopic calcified lesions were similar in the two patients, with diffuse deposits of calcium that, upon observation with polarized light, showed an amorphous to finely granular organization, with dark blue to purple aggregates without a crystalline structure. These deposits were surrounded by a chronic inflammatory infiltrate composed of lymphocytes, histiocytes, and several foreign body-type multinucleated giant cells (Fig. 3).
Genetic Testing
Genomic DNA was extracted from peripheral blood collected from the patient in Case 1 and from her daughter, grandson, and granddaughter and from the patient in Case 2 with use of the microvolume extraction method (QIAamp DNA Mini Kit; QIAGEN, Hilden, Germany), according to the manufacturer's instructions.
The three FGF23 exons, including the intron-exon boundary regions, were amplified by polymerase chain reaction with specific sets of upstream and downstream primers located in the flanking intronic regions for each exon, in a 25-µL volume containing 20 to 50 ng of DNA, 1X GoTaq Reaction Buffer (50 mM tris-HCl [pH 9.0], 50 mM NaCl, 1.5 mM MgCl2; Promega, Madison, Wisconsin), 200 µM deoxyribonucleotides, 0.4 µM of each primer, and one unit of GoTaq DNA Polymerase (Promega). After initial denaturation at 95°C for four minutes, the samples were amplified in thirty cycles of thirty seconds at 95°C, thirty seconds at the specific annealing temperature for every couple of primers, and one minute at 72°C. A final extension at 72°C for five minutes was performed. The products of the polymerase chain reaction were tested by 2% ethidium bromide-stained agarose gel electrophoresis and then purified by NucleoFast 96 PCR Plates (MACHEREY-NAGEL, Easton, Pennsylvania) for purification of the polymerase chain reaction product. Purification products were sequenced, both with forward and reverse primers, with use of the BigDye Terminator Purification Kit 3.1 (Applied Biosystems, Foster City, California) in a reaction consisting of twenty-five repeated cycles of denaturation for ten seconds at 96°C, annealing for ten seconds at 50°C, and extension for three minutes at 60°C. The sequencing products were then purified with the Montage-SEQ96 Sequencing Reaction Cleanup Kit (Millipore, Bedford, Massachusetts) and analyzed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The obtained sequences were compared with the wild-type reference sequence of the FGF23 gene published on the GenBank database (NT_009759).
For the GALNT3 gene, all ten coding exons, as well as conserved splice sites, were amplified in nine fragments by standard polymerase chain reaction methods, as indicated by Ichikawa et al.12. Polymerase chain reaction products were electrophoresed in a 2% agarose gel and purified with use of the DNA Gel Extraction Kit (QIAGEN). Approximately 100 ng of each polymerase chain reaction amplicon were directly sequenced for forward primers with use of the BigDye Terminator Purification Kit and the ABI Prism 3100 Genetic Analyzer12. No mutations of the GALNT3 gene in any of the subjects analyzed were found (data not shown).
Statistical Analysis
Statistical analysis was performed with use of the Student t test.
Source of Funding
The funding source did not play a role in the investigation.
A new homozygous H41Q, codon 41, C?A transversion at position 123 (c.123C>A) in exon 1 of the FGF23 gene was found in both affected patients (Fig. 4). An H41Q heterozygous substitution was present in the daughter of the patient in Case 1, without the identification of mutations in the patient's grandson and granddaughter. This FGF23 gene sequence variation has not been described in the SNP (single nucleotide polymorphism) databases10,13,15,16.
Moreover, in order to verify the frequency of the H41Q variant and to exclude the presence of polymorphic change, we analyzed the DNA of 100 white volunteers and did not find this change, confirming that the substitution is a novel FGF23 gene mutation. The mutation is predicted to result in the substitution of histidine for a glutamine residue at position 41 of the FGF-23 amino acid sequence. Using the ConSeq server (), we found that H41 is extremely well conserved among various homologous proteins in humans and across species (conservation scale = 8 [range, 1-9]; score = -1.349). To determine the effect of H41Q on the FGF-23 secondary structure, we used the NNPredict software17. H41Q is predicted to disrupt the ninth helix structure of the protein.
Circulating intact FGF-23 was lower in the patients with homozygous H41Q in comparison with the heterozygous and wild-type subjects. FGF-23 levels, as determined by C-terminal ELISA, were markedly elevated in both patients, while a small elevation of C-terminal FGF-23 was observed in the heterozygous daughter of the patient in Case 1 compared with the wild type. The absolute serum levels of full-length and of inactive C-terminal fragment of FGF-23 in the two patients, in the heterozygous daughter of one patient (Case 1), and in the grandchildren of the patient in Case 1 with the wild type are listed in Table II.
Calcium and phosphate intakes were similar in the two patients and in the daughter of the patient in Case 1 (Table I).
Bone mineral density in the lumbar spine and the femoral neck were reduced in both patients (0.736 g/cm2 [a lumbar spine T-score of -2.8 standard deviations and a Z score of -1.3 standard deviations] and 0.606 g/cm2 [a femoral neck T-score of -2.2 standard deviations and a Z score of -0.5 standard deviation] for Case 1, and 0.788 g/cm2 [a lumbar spine T-score of -3.2 standard deviations and a Z score of -1 standard deviations] and 0.646 g/cm2 [a femoral neck T-score of -1.9 standard deviations and a Z score of -0.9 standard deviation] for Case 2). Both patients showed normal serum calcium and parathyroid hormone levels, mild hyperphosphatemia, normal urine calcium, and inappropriately normal serum 1,25(OH)2D3. The patient in Case 1 showed a TmP/GFR of 1.67 mmol/L and the patient in Case 2, a TmP/GFR of 1.69 mmol/L (normal range, 0.71 to 1.45 mmol/L) with normal creatinine, suggesting increased Pi tubular reabsorption (Table I). In Case 1, inflammatory indices (fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) were elevated (Table I). The daughter of Case 1 showed no abnormalities in any of these biochemical parameters.
In both patients, the arterial and venous Doppler ultrasound of the lower extremity evidenced subcutaneous calcification, without identifiable lesions in the arteries and veins.