Bone 59 (2014) 114–121
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Original Full Length Article
A compound heterozygous mutation in SLC34A3 causes hereditary hypophosphatemic rickets with hypercalciuria in a Chinese patient Yue Chi a,1, Zhen Zhao a,1, Xiaodong He a, Yue Sun a, Yan Jiang a, Mei Li a, Ou Wang a, Xiaoping Xing a, Andrew Y. Sun b, Xueying Zhou a, Xunwu Meng a, Weibo Xia a,⁎ a Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Shuaifuyuan No. 1, Wangfujing, Dongcheng District, Beijing 100730, China b Harvard Medical School, Boston, MA 02115, USA
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Article history: Received 24 June 2013 Revised 4 November 2013 Accepted 10 November 2013 Available online 16 November 2013 Edited by: Bente Langdahl Keywords: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) SLC34A3 Mutation
a b s t r a c t Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder inherited in an autosomal recessive fashion and characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH was recently mapped to chromosome 9q34, which contains the gene SLC34A3 which encodes the renal proximal tubular sodium–phosphate cotransporter NaPi-IIc. Here we describe a 29-year-old man with a history of childhood rickets who presented with increased renal phosphate clearance leading to hypophosphatemia, hypercalciuria, low serum parathyroid hormone (PTH), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) and recurrent nephrolithiasis. We performed a mutation analysis of SLC34A3 (exons and adjacent introns) of the proband and his parents to determine if there was a genetic contribution. The proband proved to be compound heterozygous for two missense mutations in SLC34A3: one novel mutation in exon 7 c.571GNC (p.G191R) and one previously identified mutation in exon 13 c.1402CNT (p.R468W). His parents were both asymptomatic heterozygous carriers of one of these two mutations. We also performed an oral phosphate loading test and compared serum phosphate, intact PTH, and intact fibroblast growth factor 23 (iFGF23) in this patient versus patients with other forms of hypophosphatemic rickets, the results of which further revealed that the mechanism of hypophosphatemia in HHRH is independent of FGF23. This is the first report of HHRH in the Chinese population. Our findings of the novel mutation in exon 7 add to the list of more than 20 reported mutations of SLC34A3. © 2013 Elsevier Inc. All rights reserved.
Introduction Hereditary hypophosphatemic rickets with hypercalciuria (HHRH [MIM 241530]) is a rare, autosomal recessive disorder characterized by hypophosphatemia, short stature, rickets, and/or osteomalacia and secondary absorptive hypercalciuria. It was initially described as a new syndrome in a large, consanguineous Bedouin kindred in 1985 [1]. In HHRH, hypophosphatemia is caused by increased urinary phosphate excretion. In turn, compensatory upregulation of renal 1α-hydroxylase increases circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D), with resultant suppression of parathyroid hormone (PTH) and increased intestinal absorption of calcium resulting in an increased renal filtered calcium load and hypercalciuria. Additionally, fibroblast growth factor
⁎ Corresponding author. Fax: +86 10 6915 5076. E-mail address: [email protected] (W. Xia). 1 These authors contributed equally to this work. 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.11.008
23 (FGF23) is not elevated in HHRH, which further helps differentiate it from the FGF23-dependent hypophosphatemic disorders such as autosomal dominant hypophosphatemic rickets (ADHR; FGF23 mutation) [2], autosomal recessive hypophosphatemic rickets (ARHR; DMP1, ENPP1 mutation) [3,4], and X-linked hypophosphatemia (XLH; PHEX mutation) [5], in which FGF23 is generally high [6]. Yamamoto et al. [7] reported that the maximal increase in serum phosphate concentration after an oral phosphate loading test was significantly higher in HHRH compared to XLH, suggesting that the gastrointestinal absorption of phosphate may also be different in these two conditions [7]. The kidney is a major regulator of phosphate homeostasis [8]. In the proximal tubules of the kidney, two closely related transporters, NaPiIIa (SLC34A1) [9] and NaPi-IIc (SLC34A3) [10] reabsorb more than 70% of filtered phosphate. Bergwitz et al. [11] performed a genome-wide linkage scan combined with homozygosity mapping and mapped HHRH to the gene SLC34A3, which encodes the type IIc Na/Pi co-transporter. Recently, some homozygous and compound heterozygous mutations of SLC34A3 were reported [12,11,13]. Individuals carrying SLC34A3 mutations on both alleles display the full clinical picture of HHRH, while
Y. Chi et al. / Bone 59 (2014) 114–121 Table 1 Initial laboratory studies of the HHRH patient and his parents. Proband Mother Father Reference values Serum phosphate (mmol/L) Serum calcium (mmol/L) Serum alkaline phosphatase (U/L) Serum creatinine (μmol/L) Urine phosphate (mg/day)a Urine calcium/creatinine (mg/mg)a Urine phosphate/creatinine (mg/mg)a Tubular reabsorption of phophate (%) TmP/GFR (mg/dL) Serum intact PTH (pg/mL) Serum 25-hydroxyvitamin D (ng/mL) 1,25-dihydroxyvitamin D (pg/mL)b Serum intact FGF23 (pg/mL) Urine WBC (cells/μL)c Urine RBC (cells/μL)c Urine protein (g/L)c
0.71 2.52 219 106 794 0.22 0.62 66 1.5 5.29 10 132 19.12 500 200 1.0
1.48 2.42 119 71
1.32 2.47 76 72
12.6 12.8
17.0 13.7
0.81–1.45 2.13–2.70 30–120 53–132 700–1400 b0.20 / 75–85 N2 7–53 8–50 14.1–56.5 (29.15 ± 13.09) b15 b25 Negative
TmP/GFR mg/dL from Walton's nomogram. Abnormal results are in bold. a These urine samples were collected by full day collection. b Serum 1,25-(OH)2 vitamin D level was tested after discontinuing supplementation of 1,25-(OH)2 vitamin D for 5 days. c These urine samples were collected by fasted spot urine.
heterozygous carriers of SLC34A3 mutations often exhibit hypercalciuria, hypophosphatemia, and elevation of 1,25(OH)2D, albeit in a less pronounced manner [11]. The accurate diagnosis of HHRH has important therapeutic implications. Unlike in XLH and ADHR, phosphate supplementation alone can cause a complete remission of HHRH, whereas the addition of vitamin D can result in complications such as hypercalcemia, nephrocalcinosis, and kidney injury [12]. Here we present a patient referred to us for bone pain and recurrent nephrolithiasis. He proved to be a compound heterozygote for a novel missense mutation in exon 7 and a known missense mutation in exon 13 of SLC34A3.
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flank pain, and recurrent nephrolithiasis. The patient was unable to walk steadily at the age of three. He had bilateral genu valgum as a child and was diagnosed as “hypophosphatemic vitamin D-resistant rickets” in the local Children's Hospital. At the age of 10, renal stones were found in the right kidney by ultrasound and he subsequently underwent two operations to remove the stones. He had been treated with oral phosphate supplementation and intramuscular vitamin D from childhood until age 18. The dosage was unclear. He was also treated with calcium occasionally. He had no obvious bone pain for the duration of this treatment. After cessation of treatment, he complained of intermittent knee and left hip pain for the following ten years, which was exacerbated by prolonged sitting. Starting in the summer of 2006, the patient also complained of flank pain after prolonged sitting, accompanied by an exacerbation of his knee pain. His height had decreased approximately 3 cm from 2006 to 2008. Physical activity became restricted. Running was difficult for him and he could only walk for 1–2 h. He denied muscle weakness, dental, or hearing problems. On initial evaluation in our clinic, his height was 163 cm and his weight was 57 kg. Blood pressure was normal (110/70 mm Hg). Physical examination showed striking deformities in his chest, upper, and lower extremities. Muscle weakness was not a prominent feature. He presented with a slight rachitic rosary, prominent genu valgum, and enlargement of the wrists. Previous laboratory tests all revealed hypophosphatemia. The patient's parents were not consanguineous. The heights of the patient's father, mother and sister were 167 cm, 160 cm and 168 cm, respectively. They were all of normal stature and had no history of bone mineral disorders or nephrolithiasis. Materials and methods This study was approved by the Department of Scientific Research at Peking Union Medical College Hospital. Informed consent was obtained from the patient and his family members. Biochemical parameters
Patient presentation A 29-year-old Chinese male was referred to Peking Union Medical College Hospital in June 2008 for the evaluation of skeletal deformities,
Initial laboratory tests were done before oral phosphate supplements were administered. Fasted whole blood samples were placed at room temperature for 30 min and then centrifuged at 3000 r/min for
Fig. 1. Radiographic studies of the patient. A. X-ray of the hip and femur shows bilateral long bone bowing and general osteomalacia. B. Renal ultrasound shows multiple stones in the right kidney. C. Bone scan shows multiple areas (ribs, hips, knees, skull) of increased tracer uptake and tracer retention in the renal calices.
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Table 2 Oral phosphate loading test in patients with various forms of hypophosphatemic rickets and tumor-induced osteomalacia. 30 min
60 min
90 min
150 min
210 min
Serum phosphate (mmol/L, Mean ± 2SD) HHRH (n = 1) 0.63 XLH (n = 8) 0.59 ± 0.25 ADHR (n = 1) 0.26 TIO (n = 3) 0.4 ± 0.08 Healthy controls (n = 8) 1.31 ± 0.47
0 min
1.58 0.87 ± 0.51 0.54 0.53 ± 0.06 1.74 ± 0.59
1.84 0.90 ± 0.29 0.67 0.68 ± 0.29 1.95 ± 0.45
1.35 0.96 ± 0.26 0.54 0.69 ± 0.03 1.85 ± 0.51
1.12 0.84 ± 0.15 0.57 0.63 ± 0.18 1.67 ± 0.46
0.98 0.8 ± 0.17 0.59 0.58 ± 0.27 1.60 ± 0.41
Serum iPTH (pg/mL, Mean ± 2SD) HHRH (n = 1) 12.2 XLH (n = 8) 81.76 ± 105.1 ADHR (n = 1) 47.1 TIO (n = 3) 52.8 ± 10.3 Healthy controls (n = 8) 37.1 ± 22.8
53.8 120.42 ± 144.4 48.7 75.1 ± 19.2 51.1 ± 26.5
41.4 109.21 ± 103.3 59.7 87.5 ± 37.3 47.6 ± 38.2
35.4 104.3 ± 132.2 47.9 91.5 ± 54.1 51.2 ± 55.6
31 105.09 ± 91.7 69.2 81.9 ± 70.4 53.1 ± 40.1
27.6 104.57 ± 134.8 57.4 78.4 ± 80.6 52.4 ± 44.6
Serum iFGF23 (pg/mL, Mean ± 2SD) HHRH (n = 1) XLH (n = 8) ADHR (n = 1) TIO (n = 3) Healthy controls (n = 8)
0 min
30 min
60 min
90 min
210 min
19.1 52.2 ± 30.1 33.9 1427 ± 827.6 29.2 ± 13.1
12.1 54.1 ± 37.1 38.5 1368 ± 993 28.1 ± 8.2
17.7 52.6 ± 36.3 37.5 1369 ± 974.6 27.8 ± 6.8
12.1 55.8 ± 36.9 45.3 1471 ± 770.2 30.9 ± 9.1
19.1 52.2 ± 41.6 47.4 1428 ± 633.9 29.0 ± 6.3
3 min to separate the serum for analysis. Reference ranges were obtained from the central laboratory of PUMCH and were all age/sex/ethnically appropriate. Reference range for the FGF23 assay was calculated as the fasting mean value in 8 control patients ± 2 standard deviations. All routine laboratory studies on serum and urine were performed using routine assays available at the central laboratory of PUMCH. Serum 25hydroxyvitamin D (25(OH)D) and serum intact PTH (iPTH) levels were assayed by an automated Roche electrochemiluminescence system (E 170; Roche Diagnostics, Basel, Switzerland). Serum 1,25(OH)2D levels were determined by a 1,25-dihydroxyvitamin D 125I RIA kit (Diasorin, USA). Serum intact FGF23 (iFGF23) was measured by two-site enzyme-linked immunosorbent assay (ELISA) using an FGF23 ELISA Kit (Kainos, Japan). Both fasted spot urine and full day urine collections were obtained. Urine was analyzed by a Urinary Chemical Analyzer (Clinitek 500, SIEMENS, USA) and Urine Flow Cytometer (Sysmex UF-1000i, Sysmex, Japan) and confirmed with light microscopy. Assays were performed according to the manufacturer's protocol. The renal tubular reabsorption of phosphate was calculated using the following formula: %TRP (tubular reabsorption of phosphate) = 100 × [1 − (urine phosphate × serum creatinine) / (serum phosphate × urine creatinine). When the serum phosphate is below the reference range for age, %TRP should be above 90 [28]. TmP/GFR (tubular maximum reabsorption threshold of phosphate per glomerular filtration rate) was estimated using the Walton and Bijvoet nomogram [14,15]. Both TRP and TmP/GFR were calculated using fasting blood and urine samples.
phosphate, iPTH, and iFGF23 levels. Urine was collected from 0 min to 210 min to determine urinary phosphate excretion during this period. Patients continued to fast for the duration of the 210 min test. The test was also carried out in 8 XLH patients, 3 tumor-induced osteomalacia (TIO) patients, 1 ADHR patient, and 8 healthy Chinese volunteers. DNA-sequence analysis of SLC34A3 Genomic DNA of the proband, his parents, and a wild type control were all extracted from peripheral leukocytes using a QIAamp DNA Blood Mini Kit (50) (Qiagen, Germany). The wild type patient was chosen from a Han Chinese patient, aged 20–60 years, with normal serum phosphate and urine calcium with no history of hereditary or bone metabolism disorders. All 13 exons and 100 base pairs on both sides of the exon–intron boundaries of SLC34A3 were amplified by PCR. Primers were designed using the software Primer Premier 5.0 (Supplemental Table 1). Taq DNA polymerase (Takara, Japan) and its standard buffer were used in all reactions under the following conditions: initial denaturation at 95° for 3 min, followed by 30 cycles at 94° for 30 s, 50–58° for 30 s, and 72° for 50 s. The amplified products were sequenced by an automated sequencer (ABI3730XL) according to the manufacturer's protocol. Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information database. We used an in silico prediction method to analyze the two variants (http://genetics.bwh.harvard. edu/pph).
Oral phosphate loading test Imaging techniques To investigate gastrointestinal phosphate absorption in our HHRH patient, we performed an oral phosphate loading test after all medications were withheld for one week. The patient ingested 1.5 g of phosphate (Beijing Yanjing Pharmaceutical Co., Ltd.) given as neutral buffered sodium–potassium in 192 mL of water in the morning after fasting for at least 12 h. Venous blood samples were drawn at 30– 60 min intervals from 0 min to 210 min to determine serum
Radiographic studies were performed in the Department of Radiology at the Peking Union Medical College Hospital. Plain X-ray of the pelvis was performed to detect bony deformities. We also performed renal ultrasonography (SIEMENS, Germany) to detect renal stones. Bone scan was performed according to standard protocols with technetium-99mMDP (infinia hawkeye, GE, USA).
Fig. 2. Serum concentrations of phosphate, iPTH, and iFGF23 in patients with HHRH, XLH, ADHR, TIO, and healthy controls during an oral phosphate loading test. Dashed horizontal lines indicate the reference ranges for serum phosphate (0.81–1.45 mmol/L), iPTH (7–53 pg/mL), and intact FGF23 (29.15 ± 13.09 pg/mL) in our laboratory. All data are Mean ± 2SD. A. Serum phosphate concentrations of four studied disorders were all below the normal range at 0 min and all increased after phosphate administration. The maximal increase in serum phosphate concentration for HHRH, XLH, ADHR, TIO, and Controls were 1.21 mmol/L, 0.37 mmol/L, 0.41 mmol/L and 0.29 mmol/L, and 0.64 mmol/L, respectively. B. Serum iPTH was elevated in patients with XLH and TIO, slightly elevated in ADHR, and normal in HHRH at each time point. After administration of phosphate, iPTH levels increased in all groups. C. Serum iFGF23 concentrations were all within or above the normal range at each time point for XLH, ADHR and Controls but were consistently low or low-normal for HHRH at each time point. Slight increase in iFGF23 levels after loading was observed only in the ADHR group. Levels of iFGF23 did not significantly increase in the XLH, HHRH, and Control groups during the test.
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Fig. 3. Genetic analysis of SLC34A3 in the proband and his parents. Mutation analysis revealed a compound heterozygous mutation of SLC34A3 in the proband: c.571GNC (G191R) is a novel missense mutation in exon 7; c.1402CNT (R468W) is a previously reported missense mutation in exon 13. The proband's father and mother were both asymptomatic heterozygous carriers for c.571GNC and c.1402CNT, respectively. These two mutations were not found in the wild type.
Results Biochemical and radiographic assessment Baseline biochemical data of the patient and his parents are summarized in Table 1. Laboratory tests in the proband revealed low serum
phosphate (0.71 mmol/L), elevated alkaline phosphatase (ALP) (219 U/ L), low serum iPTH (5.29 pg/mL), and an elevated urine calcium/creatinine ratio of 0.22 mg/mg, along with normal serum calcium, creatinine, and 25(OH)D. Despite the hypophosphatemia, TRP was decreased at 66% and maximal TmP/GFR was low at 1.5 mg/dL, values consistent with renal phosphate wasting. An elevated 1,25(OH)2D level of
Fig. 4. Sites of known mutations in SLC34A3 in HHRH patients. Boxes in green/light blue indicate the 13 exons of SLC34A3. Arrows indicate sites of mutations on the exons. The two mutations found in this study are shown in blue.
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Fig. 5. Protein sequence alignment of the flanking regions of the G191R mutation among different species using BLAST®, NCBI database. The G191R mutation found in this study is in red. The amino acid glycine at position 191 (highlighted in the green column) is highly conserved among different species.
132 pg/mL was detected, which raised suspicion for HHRH. Urinalysis was notable for the presence of many red and white blood cells, and protein. The serum calcium, ALP, creatinine, iPTH, and 1,25(OH)2D of the proband's parents were all normal (Table 1). Serum phosphate was normal in the father and marginally above normal in the mother (1.48, reference range 0.81–1.45). Radiographic examination of the spine and knees (figure not shown) revealed severe generalized osteoporosis with a thin cortical layer, vertebral biconcave deformation, and features of osteomalacia. X-rays of the hip and femur showed bilateral bowing of the long bones, Looser's zones, and generalized osteomalacia (Fig. 1A). Renal ultrasound revealed multiple calculi in the right kidney (Fig. 1B) and renal cysts in the left kidney (figure not shown). Bone scan showed multiple areas of increased tracer uptake in the ribs, hips, knees, and skull, as well as tracer retention in the renal calices (Fig. 1C).
Changes in serum phosphate, iPTH, and iFGF23 in different types of hypophosphatemic rickets during an oral phosphate loading test In the oral phosphate loading test, we compared the results of our HHRH patient against patients with XLH (n = 8), TIO (n = 3), ADHR (n = 1) and healthy controls (n = 8) (Fig. 2, Table 2). Serum phosphate concentrations in patients with a form of hypophosphatemic rickets were all below the normal range at 0 min and all increased from 30 to 210 min after phosphate loading (Fig. 2A). Peak phosphate concentrations in the HHRH patient and healthy controls were both above the normal range, while the phosphate concentrations in XLH, TIO, and ADHR patients at each time point were all below or at the lower limit of normal. Maximal increase in serum phosphate for patients with HHRH, XLH, TIO, ADHR and healthy controls was 1.21 mmol/L, 0.37 mmol/L, 0.41 mmol/L and 0.29 mmol/L, and 0.64 mmol/L respectively. Serum
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iPTH was elevated in patients with XLH and TIO, slightly elevated in ADHR, and normal in HHRH at each time point (Fig. 2B). After administration of phosphate, iPTH increased in all five groups and reached peak levels at 30 min in both HHRH and XLH patients. Intact FGF23 concentrations were all within or above the normal range at each time point for XLH, ADHR and healthy controls but were consistently low or lownormal for HHRH at each time point. Increase in iFGF23 after loading was observed only in the ADHR group. Levels of iFGF23 did not significantly increase in the XLH and HHRH groups during the test. Serum iFGF23 concentrations in the TIO group were about 50-fold higher than normal (Table 2, not shown in Fig. 2). The total urinary phosphate excretion of the HHRH patient during the 210 min test was 18 mmol. Genetic analysis In the genetic analysis, a compound heterozygous mutation was found in the SLC34A3 gene of the patient. Both mutations are missense mutations. c.571GNC in exon 7 is a novel mutation which results in the replacement of glycine at position 191 by arginine (G191R). c.1402CNT in exon 13, previously identified, results in the replacement of arginine at position 468 by tryptophan (R468W) (Fig. 3). The proband's father is a carrier of c.571GNC, while his mother carries c.1402CNT, which were consistent with the mutations in their son. Both of the proband's parents were asymptomatic. The two mutations identified in this study were not found in the wild type. Discussion Here we describe a Chinese patient who presented with severe osteomalacia, hypophosphatemia, hypercalciuria, decreased TmP/GFR, elevated serum 1,25(OH)2D, and recurrent nephrolithiasis, consistent with a diagnosis of HHRH. Genetic analysis showed a compound heterozygous mutation in SLC34A3: c.571GNC in exon 7 and c.1402CNT in exon 13. HHRH is a rare autosomal recessive syndrome which was initially reported in a large consanguineous Bedouin kindred by Tieder et al. [1]. Thereafter, kindreds from other countries were reported, including Turkish [16,17], Indian [18], Belgian [11], American [19], Spanish [20], Japanese [7], Arabic [12], Iranian [21], Australian [11] and African [22]. This is the first report of HHRH in the Chinese population. All currently known disease-causing mutations of HHRH are summarized in Supplemental Table 2. The positions of the identified mutations of SLC34A3 exons are shown in Fig. 4. Our discovery of a novel mutation in exon 7 adds to the list of more than 20 mutations for this disease. SLC34A3 consists of 13 exons, with the initiation codon located in exon 2 [12]. SLC34A3 encodes the sodium–phosphate co-transport protein IIc (NaPi-IIc), a multi-pass transmembrane protein which actively transports phosphate into cells via Na+ co-transport in the renal brush border membrane [11]. Loss of function of the NaPi-IIc protein results in a primary renal tubular defect in phosphate reabsorption leading to the typical HHRH phenotype. Recent mutation analyses have implicated homozygous and compound heterozygous mutations in SLC34A3 as the genetic cause of HHRH [12,11]. Interestingly, a recent study reported that a single heterozygous mutation could cause a mild phenotype of HHRH as well [20]. In the 21 independent kindreds described to date, the mutations of the affected patients were 43% homozygous, 38% compound heterozygous, and 19% heterozygous. The mutation c.1402CNT was described by Bergwitz et al. in a patient of Turkish origin with HHRH [11]. This nucleotide change was absent in the unaffected sibling and was not identified in 350 control alleles [11]. The mutation results in the replacement of arginine at position 468 by tryptophan, a position located within the transmembrane domain of the NaPi-IIc which is evolutionarily conserved. Our in silico analysis predicted this mutation to be damaging with a probability score of 1.000 (http://genetics.bwh.harvard.edu/pph). It has been reported that heterozygosity of some missense mutations including c.1402CNT is associated with increased calcium excretion and other laboratory
abnormalities, such as mild hypophosphatemia, reduced TmP/GFR, and elevated 1,25(OH)2D [1,11,23]. This evidence indicates that the c.1402CNT mutation is likely a pathogenic variation in SLC34A3 for our patient. The proband's mutation of c.571GNC is a novel missense mutation which results in the replacement of glycine at position 191 by arginine (G191R). The amino acid G191 is in another transmembrane domain of NaPi-IIc and was predicted in silico to be probably damaging to the function of the co-transporter with a probability score of 1.000 as well (http://genetics.bwh.harvard.edu/pph). NCBI BLAST alignment results indicated that the flanking amino acid regions at the site of the G191R mutation were highly conserved among different species (Fig. 5). To investigate differences in gastrointestinal phosphate absorption, we performed an oral phosphate loading test in our patient with HHRH and compared it to patients with other forms of hypophosphatemic rickets (XLH, ADHR), patients with TIO, and healthy controls. Serum phosphate concentrations in all groups increased after taking neutral phosphate. Of the three forms of hypophosphatemic rickets studied (HHRH, XLH, ADHR), significant correction of hypophosphatemia was only observed in the HHRH patient. Maximal increase in serum phosphate in HHRH was greater than in all other groups. These findings suggest that gastrointestinal absorption of phosphate in HHRH is increased compared to XLH, ADHR, TIO, or healthy controls, likely as a result of the increased 1,25(OH)2D levels found in HHRH patients. Another hypothesis is that the hypophosphatemic diseases where the phosphaturic factor FGF23 is elevated demonstrate greater renal phosphate excretion compared to HHRH, which typically presents with low FGF23 levels. These findings are consistent with observations by Yamamoto et al. in Japanese patients [7]. In their study, they also found that the maximal increase in serum phosphate levels and peak serum phosphate concentration were significantly higher in HHRH patients compared to XLH patients. Concurrently, we observed that after administration of phosphate, iPTH levels increased in the five groups, indicating that PTH is stimulated after correction of the hypophosphatemia. However, PTH levels are primarily controlled by serum calcium levels. We also observed that PTH levels in XLH, ADHR, and TIO patients were significantly higher than in the HHRH patient, which in turn can be explained by the different serum calcium levels in HHRH versus the FGF23-dependent forms of hypophosphatemic rickets. Additionally, we measured serum concentrations of the phosphaturic factor FGF23 during the oral phosphate loading test. Levels of iFGF23 in the HHRH patient were much lower than those in all other groups and were around the lower limit of normal throughout the phosphate loading test. Previous studies have shown that FGF23 is frequently elevated in patients with TIO and XLH, but not universally elevated in patients with ADHR [24], findings consistent with our work. Lorenz-Depiereux et al. [12] reported that FGF23 is at normal or low-normal serum levels in HHRH patients, which we also observed in our patient throughout the phosphate loading test. These findings show that the mechanism of hypophosphatemia in HHRH is independent of FGF23. Interestingly, as phosphate is a driver of FGF23, one might expect a transient increase in FGF23 in all groups after phosphate loading, but this was not seen. This adds to previous evidence showing that oral phosphate loading does not rapidly increase serum FGF23 levels in healthy men [25]. The patient's radiographic studies and prior surgeries reveal a longstanding history of severe nephrolithiasis. It has been reported that despite similar biochemical changes seen in heterozygous and homozygous carriers of SLC34A3 mutations, hypercalciuria does not lead to nephrolithiasis or nephrocalcinosis unless the affected individuals are inappropriately treated with vitamin D analogs [26,27]. Indeed, our patient had been treated with phosphate, calcium and 1,25(OH)2D supplementation for many years before the diagnosis of HHRH was established. Interestingly, some studies raise a possibility that some NaPi-IIc mutations such as g.4225_50del cause a dominant form of nephrolithiasis [16,17], but this hypothesis has not yet been proven. Further investigations are required to determine whether certain NaPi-IIc mutations are associated with renal calcification.
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An early and accurate diagnosis of HHRH has important therapeutic implications. Inorganic phosphate supplementation alone can cause a complete remission of HHRH, whereas the addition of vitamin D can create complications such as hypercalcemia, nephrocalcinosis, and renal damage. Nephrocalcinosis or nephrolithiasis can be the first symptoms of HHRH in patients who were mistreated with vitamin D and calcium, and therefore NaPi-IIc mutations should be considered in the differential diagnosis when these kidney findings are observed in the appropriate clinical context [17]. Hypercalciuria is frequent in the family members of HHRH patients. Thus, HHRH may be an underdiagnosed condition, mislabeled as idiopathic hypercalciuria or osteopenia. Furthermore, in Kremke et al.'s longitudinal follow-up study, they illustrated that vitamin D deficiency can mask some of the characteristic laboratory findings of HHRH. Thus, vitamin D levels should be checked and renal ultrasonography should be performed when considering the diagnosis of HHRH. Biochemical assessment and renal ultrasounds are also recommended for first-degree relatives of HHRH patients, especially when asymptomatic renal stones are present. There are some limitations in our study. We did not conduct a functional analysis of the mutant NaPi-IIc protein of our patient. Further studies are needed to determine the accurate function of NaPi-IIc in maintaining phosphate homeostasis. Conclusions Here we report the first case of HHRH in the Chinese population. The patient proved to be a compound heterozygote for a novel missense mutation and a previously described missense mutation of the gene SLC34A3. The discovery of this novel mutation adds to the list of more than 20 mutations for this disease. Results from an oral phosphate loading test further reveal that the mechanism of hypophosphatemia in HHRH is independent of FGF23. Disclosure page The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (No.81070687 and 81170805), National Science and Technology Pillar Program (2006BAI02B03), National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (Grant2008ZX09312-016), Beijing Natural Science Foundation (No. 7121012) and Scientific Research Foundation of Beijing Medical Development (No. 2007-3029). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2013.11.008. References [1] Tieder M, Modai D, Samuel R, Arie R, Halabe A, Bab I, et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985;312:611–7. [2] Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345–8.
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[3] Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 2006;38:1310–5. [4] Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, Chalifa-Caspi V, et al. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 2010;86(2):273–8. [5] A gene (PEX) with homologies to endopeptidases is mutated in patients with Xlincked hypophosphatemic rickets. The HYP Consortium. Nat Genet 1995;11:130–6. [6] Seiji F, Yuichiro S. Fibroblast growth factor 23 as a phosphotropic hormone and beyond. J Bone Miner Metab 2011;29:507–14. [7] Yamamoto T, Michigami T, Aranami F, Segawa H, Yoh K, Nakajima S, et al. Hereditary hypophosphatemic rickets with hypercalciuria: a study for the phosphate transporter gene type IIc and osteoblastic function. J Bone Miner Metab 2007;25:407–13. [8] Tenenhouse HS. Cellular and molecular mechanisms of renal phosphate transport. J Bone Miner Res 1997;12:159–64. [9] Magagnin S, Werner A, Markovich D, Sorribas V, Stange G, Biber J, et al. Expression cloning of human and rat renal cortex Na/Pi cotransport. Proc Natl Acad Sci U S A 1993;90:5979–83. [10] Segawa H, Kaneko I, Takahashi A, Kuwahata M, Ito M, Ohkido I, et al. Growth-related renal type II Na/Pi cotransporter. J Biol Chem 2002;277:19665–72. [11] Bergwitz C, Roslin NM, Tieder M, Loredo-Osti JC, Bastepe M, Abu-Zahra H, et al. SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium–phosphate cotransporter NaP(i)-IIc in maintaining phosphate homeostasis. Am J Hum Genet 2006;78:179–92. [12] Lorenz-Depiereux B, Benet-Pages A, Eckstein G, Tenenbaum-Rakover Y, Wagenstaller J, Tiosano D, et al. Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium–phosphate cotransporter gene SLC34A3. Am J Hum Genet 2006;78:193–201. [13] Ichikawa S, Sorenson AH, Imel EA, Friedman NE, Gertner JM, Econs MJ. Intronic deletions in the SLC34A3 gene cause hereditary hypophosphatemic rickets with hypercalciuria. J Clin Endocrinol Metab 2006;91:4022–7. [14] Walton RJ, Bijvoet OL. Nomogram for derivation of renal threshold phosphate concentration. Lancet 1975;2:309–10. [15] Brodehl J, Krause A, Hoyer PF. Assessment of maximal tubular phosphate reabsorption: comparison of direct measurement with the nomogram of Bijvoet. Pediatr Nephrol 1988;2:183–9. [16] Jaureguiberry G, Carpenter TO, Forman S, Jüppner H, Bergwitz C. A novel missense mutation in SLC34A3 that cuases hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium–phosphate cotransport in NaPi-IIc. Am J Physiol Renal Physiol 2008;295:F371–9. [17] Kremke B, Bergwitz C, Ahrens W, Schütt S, Schumacher M, Wagner V, et al. Hypophosphatemic rickets with hypercalciuria due to mutation in SLC34A3/ NaPi-IIc can be masked by vitamin D deficiency and can be associated with renal calcifications. Exp Clin Endocrinol Diabetes 2009;117:49–56. [18] Page K, Bergwitz C, Jaureguiberry G, Harinarayan CV, Insogna K. A patient with hypophosphatemia, a femoral fracture and recurrent kidney stones — report of a novel mutation in SLC34A3 (p.Q49X). Endocr Pract 2008;14(7):869–74. [19] Yu Y, Sanderson SR, Reyes M, Sharma A, Dunbar N, Srivastava T, et al. Novel NaPi-IIc mutations causing HHRH and idiopathic hypercalciuria in several unrelated families: long-term follow-up in one kindred. Bone 2012;50:1100–6. [20] Natalia MG, Helena GP, Eliecer C, Teresa MP, Fernando S. Genetic and clinical peculiarities in a new family with hereditary hypophosphatemic rickets with hypercalciuria: a case report. Orphanet J Rare Dis 2010;5:1. [21] Shirin HR, Mahsa MA, Azadeh EH, Ehsan D, Akbar S, Parvin A, et al. SLC34A3 intronic deletion in a new kindred with hereditary hypophosphatemic rickets with hypercalciuria. J Clin Res Pediatr Endocrinol 2012;4(2):89–93. [22] Braithwaite V, Pettifor JM, Prentice A. Novel SLC34A3 mutation causing hereditary hypophosphatemic rickets with hypercalciuria in a Gambian family. Bone 2012;53(1):216–20. [23] Tieder M, Modai D, Shaked U, Samuel R, Arie R, Halabe A, et al. ‘Idiopathic’ hypercalciuria and hereditary hypophosphatemic rickets. Two phenotypical expression of a common genetic defect. N Engl J Med 1987;316:125–9. [24] Imel Eui SL, Econs MJ. FGF23 concentrations vary with disease status in autosomal dominant hypophosphatemic rickets. J Bone Miner Res 2007;22(4):520–6. [25] Nishida Y, Taketani Y, Yamanaka-Okumura H, Imamura F, Taniguchi A. Acute effect of oral phosphate loading on serum fibroblast growth factor 23 levels in healthy men. Kidney Int 2006;70:2141–7. [26] Chen C, Carpenter T, Steg N, Baron R, Anast C. Hypercalciuric hypophosphatemic rickets, mineral balance, bone histomorphometry and therapeutic implications of hypercalciuria. Pediatrics 1989;84:276–80. [27] Tieder M, Blonder J, Strauss S, Shaked U, Maor J, Gabizon D, et al. Hyperoxaluria is not a cause of nephrocalcinosis in phosphate-treated patients with hereditary hypophosphatemic rickets. Nephron 1993;64:526–31. [28] Areses-Trapote R, López-Garcia JA, Ubetagoyena-Arrieta M, Eizaguirre A, Sáez-Villaverde R. Hereditary hypophosphatemic rickets with hypercalciuria: a case report. Nefrologia 2012;32(4):529–34.
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Original Full Length Article
A compound heterozygous mutation in SLC34A3 causes hereditary hypophosphatemic rickets with hypercalciuria in a Chinese patient Yue Chi a,1, Zhen Zhao a,1, Xiaodong He a, Yue Sun a, Yan Jiang a, Mei Li a, Ou Wang a, Xiaoping Xing a, Andrew Y. Sun b, Xueying Zhou a, Xunwu Meng a, Weibo Xia a,⁎ a Department of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Shuaifuyuan No. 1, Wangfujing, Dongcheng District, Beijing 100730, China b Harvard Medical School, Boston, MA 02115, USA
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Article history: Received 24 June 2013 Revised 4 November 2013 Accepted 10 November 2013 Available online 16 November 2013 Edited by: Bente Langdahl Keywords: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) SLC34A3 Mutation
a b s t r a c t Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare metabolic disorder inherited in an autosomal recessive fashion and characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH was recently mapped to chromosome 9q34, which contains the gene SLC34A3 which encodes the renal proximal tubular sodium–phosphate cotransporter NaPi-IIc. Here we describe a 29-year-old man with a history of childhood rickets who presented with increased renal phosphate clearance leading to hypophosphatemia, hypercalciuria, low serum parathyroid hormone (PTH), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) and recurrent nephrolithiasis. We performed a mutation analysis of SLC34A3 (exons and adjacent introns) of the proband and his parents to determine if there was a genetic contribution. The proband proved to be compound heterozygous for two missense mutations in SLC34A3: one novel mutation in exon 7 c.571GNC (p.G191R) and one previously identified mutation in exon 13 c.1402CNT (p.R468W). His parents were both asymptomatic heterozygous carriers of one of these two mutations. We also performed an oral phosphate loading test and compared serum phosphate, intact PTH, and intact fibroblast growth factor 23 (iFGF23) in this patient versus patients with other forms of hypophosphatemic rickets, the results of which further revealed that the mechanism of hypophosphatemia in HHRH is independent of FGF23. This is the first report of HHRH in the Chinese population. Our findings of the novel mutation in exon 7 add to the list of more than 20 reported mutations of SLC34A3. © 2013 Elsevier Inc. All rights reserved.
Introduction Hereditary hypophosphatemic rickets with hypercalciuria (HHRH [MIM 241530]) is a rare, autosomal recessive disorder characterized by hypophosphatemia, short stature, rickets, and/or osteomalacia and secondary absorptive hypercalciuria. It was initially described as a new syndrome in a large, consanguineous Bedouin kindred in 1985 [1]. In HHRH, hypophosphatemia is caused by increased urinary phosphate excretion. In turn, compensatory upregulation of renal 1α-hydroxylase increases circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D), with resultant suppression of parathyroid hormone (PTH) and increased intestinal absorption of calcium resulting in an increased renal filtered calcium load and hypercalciuria. Additionally, fibroblast growth factor
⁎ Corresponding author. Fax: +86 10 6915 5076. E-mail address: [email protected] (W. Xia). 1 These authors contributed equally to this work. 8756-3282/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bone.2013.11.008
23 (FGF23) is not elevated in HHRH, which further helps differentiate it from the FGF23-dependent hypophosphatemic disorders such as autosomal dominant hypophosphatemic rickets (ADHR; FGF23 mutation) [2], autosomal recessive hypophosphatemic rickets (ARHR; DMP1, ENPP1 mutation) [3,4], and X-linked hypophosphatemia (XLH; PHEX mutation) [5], in which FGF23 is generally high [6]. Yamamoto et al. [7] reported that the maximal increase in serum phosphate concentration after an oral phosphate loading test was significantly higher in HHRH compared to XLH, suggesting that the gastrointestinal absorption of phosphate may also be different in these two conditions [7]. The kidney is a major regulator of phosphate homeostasis [8]. In the proximal tubules of the kidney, two closely related transporters, NaPiIIa (SLC34A1) [9] and NaPi-IIc (SLC34A3) [10] reabsorb more than 70% of filtered phosphate. Bergwitz et al. [11] performed a genome-wide linkage scan combined with homozygosity mapping and mapped HHRH to the gene SLC34A3, which encodes the type IIc Na/Pi co-transporter. Recently, some homozygous and compound heterozygous mutations of SLC34A3 were reported [12,11,13]. Individuals carrying SLC34A3 mutations on both alleles display the full clinical picture of HHRH, while
Y. Chi et al. / Bone 59 (2014) 114–121 Table 1 Initial laboratory studies of the HHRH patient and his parents. Proband Mother Father Reference values Serum phosphate (mmol/L) Serum calcium (mmol/L) Serum alkaline phosphatase (U/L) Serum creatinine (μmol/L) Urine phosphate (mg/day)a Urine calcium/creatinine (mg/mg)a Urine phosphate/creatinine (mg/mg)a Tubular reabsorption of phophate (%) TmP/GFR (mg/dL) Serum intact PTH (pg/mL) Serum 25-hydroxyvitamin D (ng/mL) 1,25-dihydroxyvitamin D (pg/mL)b Serum intact FGF23 (pg/mL) Urine WBC (cells/μL)c Urine RBC (cells/μL)c Urine protein (g/L)c
0.71 2.52 219 106 794 0.22 0.62 66 1.5 5.29 10 132 19.12 500 200 1.0
1.48 2.42 119 71
1.32 2.47 76 72
12.6 12.8
17.0 13.7
0.81–1.45 2.13–2.70 30–120 53–132 700–1400 b0.20 / 75–85 N2 7–53 8–50 14.1–56.5 (29.15 ± 13.09) b15 b25 Negative
TmP/GFR mg/dL from Walton's nomogram. Abnormal results are in bold. a These urine samples were collected by full day collection. b Serum 1,25-(OH)2 vitamin D level was tested after discontinuing supplementation of 1,25-(OH)2 vitamin D for 5 days. c These urine samples were collected by fasted spot urine.
heterozygous carriers of SLC34A3 mutations often exhibit hypercalciuria, hypophosphatemia, and elevation of 1,25(OH)2D, albeit in a less pronounced manner [11]. The accurate diagnosis of HHRH has important therapeutic implications. Unlike in XLH and ADHR, phosphate supplementation alone can cause a complete remission of HHRH, whereas the addition of vitamin D can result in complications such as hypercalcemia, nephrocalcinosis, and kidney injury [12]. Here we present a patient referred to us for bone pain and recurrent nephrolithiasis. He proved to be a compound heterozygote for a novel missense mutation in exon 7 and a known missense mutation in exon 13 of SLC34A3.
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flank pain, and recurrent nephrolithiasis. The patient was unable to walk steadily at the age of three. He had bilateral genu valgum as a child and was diagnosed as “hypophosphatemic vitamin D-resistant rickets” in the local Children's Hospital. At the age of 10, renal stones were found in the right kidney by ultrasound and he subsequently underwent two operations to remove the stones. He had been treated with oral phosphate supplementation and intramuscular vitamin D from childhood until age 18. The dosage was unclear. He was also treated with calcium occasionally. He had no obvious bone pain for the duration of this treatment. After cessation of treatment, he complained of intermittent knee and left hip pain for the following ten years, which was exacerbated by prolonged sitting. Starting in the summer of 2006, the patient also complained of flank pain after prolonged sitting, accompanied by an exacerbation of his knee pain. His height had decreased approximately 3 cm from 2006 to 2008. Physical activity became restricted. Running was difficult for him and he could only walk for 1–2 h. He denied muscle weakness, dental, or hearing problems. On initial evaluation in our clinic, his height was 163 cm and his weight was 57 kg. Blood pressure was normal (110/70 mm Hg). Physical examination showed striking deformities in his chest, upper, and lower extremities. Muscle weakness was not a prominent feature. He presented with a slight rachitic rosary, prominent genu valgum, and enlargement of the wrists. Previous laboratory tests all revealed hypophosphatemia. The patient's parents were not consanguineous. The heights of the patient's father, mother and sister were 167 cm, 160 cm and 168 cm, respectively. They were all of normal stature and had no history of bone mineral disorders or nephrolithiasis. Materials and methods This study was approved by the Department of Scientific Research at Peking Union Medical College Hospital. Informed consent was obtained from the patient and his family members. Biochemical parameters
Patient presentation A 29-year-old Chinese male was referred to Peking Union Medical College Hospital in June 2008 for the evaluation of skeletal deformities,
Initial laboratory tests were done before oral phosphate supplements were administered. Fasted whole blood samples were placed at room temperature for 30 min and then centrifuged at 3000 r/min for
Fig. 1. Radiographic studies of the patient. A. X-ray of the hip and femur shows bilateral long bone bowing and general osteomalacia. B. Renal ultrasound shows multiple stones in the right kidney. C. Bone scan shows multiple areas (ribs, hips, knees, skull) of increased tracer uptake and tracer retention in the renal calices.
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Table 2 Oral phosphate loading test in patients with various forms of hypophosphatemic rickets and tumor-induced osteomalacia. 30 min
60 min
90 min
150 min
210 min
Serum phosphate (mmol/L, Mean ± 2SD) HHRH (n = 1) 0.63 XLH (n = 8) 0.59 ± 0.25 ADHR (n = 1) 0.26 TIO (n = 3) 0.4 ± 0.08 Healthy controls (n = 8) 1.31 ± 0.47
0 min
1.58 0.87 ± 0.51 0.54 0.53 ± 0.06 1.74 ± 0.59
1.84 0.90 ± 0.29 0.67 0.68 ± 0.29 1.95 ± 0.45
1.35 0.96 ± 0.26 0.54 0.69 ± 0.03 1.85 ± 0.51
1.12 0.84 ± 0.15 0.57 0.63 ± 0.18 1.67 ± 0.46
0.98 0.8 ± 0.17 0.59 0.58 ± 0.27 1.60 ± 0.41
Serum iPTH (pg/mL, Mean ± 2SD) HHRH (n = 1) 12.2 XLH (n = 8) 81.76 ± 105.1 ADHR (n = 1) 47.1 TIO (n = 3) 52.8 ± 10.3 Healthy controls (n = 8) 37.1 ± 22.8
53.8 120.42 ± 144.4 48.7 75.1 ± 19.2 51.1 ± 26.5
41.4 109.21 ± 103.3 59.7 87.5 ± 37.3 47.6 ± 38.2
35.4 104.3 ± 132.2 47.9 91.5 ± 54.1 51.2 ± 55.6
31 105.09 ± 91.7 69.2 81.9 ± 70.4 53.1 ± 40.1
27.6 104.57 ± 134.8 57.4 78.4 ± 80.6 52.4 ± 44.6
Serum iFGF23 (pg/mL, Mean ± 2SD) HHRH (n = 1) XLH (n = 8) ADHR (n = 1) TIO (n = 3) Healthy controls (n = 8)
0 min
30 min
60 min
90 min
210 min
19.1 52.2 ± 30.1 33.9 1427 ± 827.6 29.2 ± 13.1
12.1 54.1 ± 37.1 38.5 1368 ± 993 28.1 ± 8.2
17.7 52.6 ± 36.3 37.5 1369 ± 974.6 27.8 ± 6.8
12.1 55.8 ± 36.9 45.3 1471 ± 770.2 30.9 ± 9.1
19.1 52.2 ± 41.6 47.4 1428 ± 633.9 29.0 ± 6.3
3 min to separate the serum for analysis. Reference ranges were obtained from the central laboratory of PUMCH and were all age/sex/ethnically appropriate. Reference range for the FGF23 assay was calculated as the fasting mean value in 8 control patients ± 2 standard deviations. All routine laboratory studies on serum and urine were performed using routine assays available at the central laboratory of PUMCH. Serum 25hydroxyvitamin D (25(OH)D) and serum intact PTH (iPTH) levels were assayed by an automated Roche electrochemiluminescence system (E 170; Roche Diagnostics, Basel, Switzerland). Serum 1,25(OH)2D levels were determined by a 1,25-dihydroxyvitamin D 125I RIA kit (Diasorin, USA). Serum intact FGF23 (iFGF23) was measured by two-site enzyme-linked immunosorbent assay (ELISA) using an FGF23 ELISA Kit (Kainos, Japan). Both fasted spot urine and full day urine collections were obtained. Urine was analyzed by a Urinary Chemical Analyzer (Clinitek 500, SIEMENS, USA) and Urine Flow Cytometer (Sysmex UF-1000i, Sysmex, Japan) and confirmed with light microscopy. Assays were performed according to the manufacturer's protocol. The renal tubular reabsorption of phosphate was calculated using the following formula: %TRP (tubular reabsorption of phosphate) = 100 × [1 − (urine phosphate × serum creatinine) / (serum phosphate × urine creatinine). When the serum phosphate is below the reference range for age, %TRP should be above 90 [28]. TmP/GFR (tubular maximum reabsorption threshold of phosphate per glomerular filtration rate) was estimated using the Walton and Bijvoet nomogram [14,15]. Both TRP and TmP/GFR were calculated using fasting blood and urine samples.
phosphate, iPTH, and iFGF23 levels. Urine was collected from 0 min to 210 min to determine urinary phosphate excretion during this period. Patients continued to fast for the duration of the 210 min test. The test was also carried out in 8 XLH patients, 3 tumor-induced osteomalacia (TIO) patients, 1 ADHR patient, and 8 healthy Chinese volunteers. DNA-sequence analysis of SLC34A3 Genomic DNA of the proband, his parents, and a wild type control were all extracted from peripheral leukocytes using a QIAamp DNA Blood Mini Kit (50) (Qiagen, Germany). The wild type patient was chosen from a Han Chinese patient, aged 20–60 years, with normal serum phosphate and urine calcium with no history of hereditary or bone metabolism disorders. All 13 exons and 100 base pairs on both sides of the exon–intron boundaries of SLC34A3 were amplified by PCR. Primers were designed using the software Primer Premier 5.0 (Supplemental Table 1). Taq DNA polymerase (Takara, Japan) and its standard buffer were used in all reactions under the following conditions: initial denaturation at 95° for 3 min, followed by 30 cycles at 94° for 30 s, 50–58° for 30 s, and 72° for 50 s. The amplified products were sequenced by an automated sequencer (ABI3730XL) according to the manufacturer's protocol. Sequence alignment was performed using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information database. We used an in silico prediction method to analyze the two variants (http://genetics.bwh.harvard. edu/pph).
Oral phosphate loading test Imaging techniques To investigate gastrointestinal phosphate absorption in our HHRH patient, we performed an oral phosphate loading test after all medications were withheld for one week. The patient ingested 1.5 g of phosphate (Beijing Yanjing Pharmaceutical Co., Ltd.) given as neutral buffered sodium–potassium in 192 mL of water in the morning after fasting for at least 12 h. Venous blood samples were drawn at 30– 60 min intervals from 0 min to 210 min to determine serum
Radiographic studies were performed in the Department of Radiology at the Peking Union Medical College Hospital. Plain X-ray of the pelvis was performed to detect bony deformities. We also performed renal ultrasonography (SIEMENS, Germany) to detect renal stones. Bone scan was performed according to standard protocols with technetium-99mMDP (infinia hawkeye, GE, USA).
Fig. 2. Serum concentrations of phosphate, iPTH, and iFGF23 in patients with HHRH, XLH, ADHR, TIO, and healthy controls during an oral phosphate loading test. Dashed horizontal lines indicate the reference ranges for serum phosphate (0.81–1.45 mmol/L), iPTH (7–53 pg/mL), and intact FGF23 (29.15 ± 13.09 pg/mL) in our laboratory. All data are Mean ± 2SD. A. Serum phosphate concentrations of four studied disorders were all below the normal range at 0 min and all increased after phosphate administration. The maximal increase in serum phosphate concentration for HHRH, XLH, ADHR, TIO, and Controls were 1.21 mmol/L, 0.37 mmol/L, 0.41 mmol/L and 0.29 mmol/L, and 0.64 mmol/L, respectively. B. Serum iPTH was elevated in patients with XLH and TIO, slightly elevated in ADHR, and normal in HHRH at each time point. After administration of phosphate, iPTH levels increased in all groups. C. Serum iFGF23 concentrations were all within or above the normal range at each time point for XLH, ADHR and Controls but were consistently low or low-normal for HHRH at each time point. Slight increase in iFGF23 levels after loading was observed only in the ADHR group. Levels of iFGF23 did not significantly increase in the XLH, HHRH, and Control groups during the test.
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Fig. 3. Genetic analysis of SLC34A3 in the proband and his parents. Mutation analysis revealed a compound heterozygous mutation of SLC34A3 in the proband: c.571GNC (G191R) is a novel missense mutation in exon 7; c.1402CNT (R468W) is a previously reported missense mutation in exon 13. The proband's father and mother were both asymptomatic heterozygous carriers for c.571GNC and c.1402CNT, respectively. These two mutations were not found in the wild type.
Results Biochemical and radiographic assessment Baseline biochemical data of the patient and his parents are summarized in Table 1. Laboratory tests in the proband revealed low serum
phosphate (0.71 mmol/L), elevated alkaline phosphatase (ALP) (219 U/ L), low serum iPTH (5.29 pg/mL), and an elevated urine calcium/creatinine ratio of 0.22 mg/mg, along with normal serum calcium, creatinine, and 25(OH)D. Despite the hypophosphatemia, TRP was decreased at 66% and maximal TmP/GFR was low at 1.5 mg/dL, values consistent with renal phosphate wasting. An elevated 1,25(OH)2D level of
Fig. 4. Sites of known mutations in SLC34A3 in HHRH patients. Boxes in green/light blue indicate the 13 exons of SLC34A3. Arrows indicate sites of mutations on the exons. The two mutations found in this study are shown in blue.
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Fig. 5. Protein sequence alignment of the flanking regions of the G191R mutation among different species using BLAST®, NCBI database. The G191R mutation found in this study is in red. The amino acid glycine at position 191 (highlighted in the green column) is highly conserved among different species.
132 pg/mL was detected, which raised suspicion for HHRH. Urinalysis was notable for the presence of many red and white blood cells, and protein. The serum calcium, ALP, creatinine, iPTH, and 1,25(OH)2D of the proband's parents were all normal (Table 1). Serum phosphate was normal in the father and marginally above normal in the mother (1.48, reference range 0.81–1.45). Radiographic examination of the spine and knees (figure not shown) revealed severe generalized osteoporosis with a thin cortical layer, vertebral biconcave deformation, and features of osteomalacia. X-rays of the hip and femur showed bilateral bowing of the long bones, Looser's zones, and generalized osteomalacia (Fig. 1A). Renal ultrasound revealed multiple calculi in the right kidney (Fig. 1B) and renal cysts in the left kidney (figure not shown). Bone scan showed multiple areas of increased tracer uptake in the ribs, hips, knees, and skull, as well as tracer retention in the renal calices (Fig. 1C).
Changes in serum phosphate, iPTH, and iFGF23 in different types of hypophosphatemic rickets during an oral phosphate loading test In the oral phosphate loading test, we compared the results of our HHRH patient against patients with XLH (n = 8), TIO (n = 3), ADHR (n = 1) and healthy controls (n = 8) (Fig. 2, Table 2). Serum phosphate concentrations in patients with a form of hypophosphatemic rickets were all below the normal range at 0 min and all increased from 30 to 210 min after phosphate loading (Fig. 2A). Peak phosphate concentrations in the HHRH patient and healthy controls were both above the normal range, while the phosphate concentrations in XLH, TIO, and ADHR patients at each time point were all below or at the lower limit of normal. Maximal increase in serum phosphate for patients with HHRH, XLH, TIO, ADHR and healthy controls was 1.21 mmol/L, 0.37 mmol/L, 0.41 mmol/L and 0.29 mmol/L, and 0.64 mmol/L respectively. Serum
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iPTH was elevated in patients with XLH and TIO, slightly elevated in ADHR, and normal in HHRH at each time point (Fig. 2B). After administration of phosphate, iPTH increased in all five groups and reached peak levels at 30 min in both HHRH and XLH patients. Intact FGF23 concentrations were all within or above the normal range at each time point for XLH, ADHR and healthy controls but were consistently low or lownormal for HHRH at each time point. Increase in iFGF23 after loading was observed only in the ADHR group. Levels of iFGF23 did not significantly increase in the XLH and HHRH groups during the test. Serum iFGF23 concentrations in the TIO group were about 50-fold higher than normal (Table 2, not shown in Fig. 2). The total urinary phosphate excretion of the HHRH patient during the 210 min test was 18 mmol. Genetic analysis In the genetic analysis, a compound heterozygous mutation was found in the SLC34A3 gene of the patient. Both mutations are missense mutations. c.571GNC in exon 7 is a novel mutation which results in the replacement of glycine at position 191 by arginine (G191R). c.1402CNT in exon 13, previously identified, results in the replacement of arginine at position 468 by tryptophan (R468W) (Fig. 3). The proband's father is a carrier of c.571GNC, while his mother carries c.1402CNT, which were consistent with the mutations in their son. Both of the proband's parents were asymptomatic. The two mutations identified in this study were not found in the wild type. Discussion Here we describe a Chinese patient who presented with severe osteomalacia, hypophosphatemia, hypercalciuria, decreased TmP/GFR, elevated serum 1,25(OH)2D, and recurrent nephrolithiasis, consistent with a diagnosis of HHRH. Genetic analysis showed a compound heterozygous mutation in SLC34A3: c.571GNC in exon 7 and c.1402CNT in exon 13. HHRH is a rare autosomal recessive syndrome which was initially reported in a large consanguineous Bedouin kindred by Tieder et al. [1]. Thereafter, kindreds from other countries were reported, including Turkish [16,17], Indian [18], Belgian [11], American [19], Spanish [20], Japanese [7], Arabic [12], Iranian [21], Australian [11] and African [22]. This is the first report of HHRH in the Chinese population. All currently known disease-causing mutations of HHRH are summarized in Supplemental Table 2. The positions of the identified mutations of SLC34A3 exons are shown in Fig. 4. Our discovery of a novel mutation in exon 7 adds to the list of more than 20 mutations for this disease. SLC34A3 consists of 13 exons, with the initiation codon located in exon 2 [12]. SLC34A3 encodes the sodium–phosphate co-transport protein IIc (NaPi-IIc), a multi-pass transmembrane protein which actively transports phosphate into cells via Na+ co-transport in the renal brush border membrane [11]. Loss of function of the NaPi-IIc protein results in a primary renal tubular defect in phosphate reabsorption leading to the typical HHRH phenotype. Recent mutation analyses have implicated homozygous and compound heterozygous mutations in SLC34A3 as the genetic cause of HHRH [12,11]. Interestingly, a recent study reported that a single heterozygous mutation could cause a mild phenotype of HHRH as well [20]. In the 21 independent kindreds described to date, the mutations of the affected patients were 43% homozygous, 38% compound heterozygous, and 19% heterozygous. The mutation c.1402CNT was described by Bergwitz et al. in a patient of Turkish origin with HHRH [11]. This nucleotide change was absent in the unaffected sibling and was not identified in 350 control alleles [11]. The mutation results in the replacement of arginine at position 468 by tryptophan, a position located within the transmembrane domain of the NaPi-IIc which is evolutionarily conserved. Our in silico analysis predicted this mutation to be damaging with a probability score of 1.000 (http://genetics.bwh.harvard.edu/pph). It has been reported that heterozygosity of some missense mutations including c.1402CNT is associated with increased calcium excretion and other laboratory
abnormalities, such as mild hypophosphatemia, reduced TmP/GFR, and elevated 1,25(OH)2D [1,11,23]. This evidence indicates that the c.1402CNT mutation is likely a pathogenic variation in SLC34A3 for our patient. The proband's mutation of c.571GNC is a novel missense mutation which results in the replacement of glycine at position 191 by arginine (G191R). The amino acid G191 is in another transmembrane domain of NaPi-IIc and was predicted in silico to be probably damaging to the function of the co-transporter with a probability score of 1.000 as well (http://genetics.bwh.harvard.edu/pph). NCBI BLAST alignment results indicated that the flanking amino acid regions at the site of the G191R mutation were highly conserved among different species (Fig. 5). To investigate differences in gastrointestinal phosphate absorption, we performed an oral phosphate loading test in our patient with HHRH and compared it to patients with other forms of hypophosphatemic rickets (XLH, ADHR), patients with TIO, and healthy controls. Serum phosphate concentrations in all groups increased after taking neutral phosphate. Of the three forms of hypophosphatemic rickets studied (HHRH, XLH, ADHR), significant correction of hypophosphatemia was only observed in the HHRH patient. Maximal increase in serum phosphate in HHRH was greater than in all other groups. These findings suggest that gastrointestinal absorption of phosphate in HHRH is increased compared to XLH, ADHR, TIO, or healthy controls, likely as a result of the increased 1,25(OH)2D levels found in HHRH patients. Another hypothesis is that the hypophosphatemic diseases where the phosphaturic factor FGF23 is elevated demonstrate greater renal phosphate excretion compared to HHRH, which typically presents with low FGF23 levels. These findings are consistent with observations by Yamamoto et al. in Japanese patients [7]. In their study, they also found that the maximal increase in serum phosphate levels and peak serum phosphate concentration were significantly higher in HHRH patients compared to XLH patients. Concurrently, we observed that after administration of phosphate, iPTH levels increased in the five groups, indicating that PTH is stimulated after correction of the hypophosphatemia. However, PTH levels are primarily controlled by serum calcium levels. We also observed that PTH levels in XLH, ADHR, and TIO patients were significantly higher than in the HHRH patient, which in turn can be explained by the different serum calcium levels in HHRH versus the FGF23-dependent forms of hypophosphatemic rickets. Additionally, we measured serum concentrations of the phosphaturic factor FGF23 during the oral phosphate loading test. Levels of iFGF23 in the HHRH patient were much lower than those in all other groups and were around the lower limit of normal throughout the phosphate loading test. Previous studies have shown that FGF23 is frequently elevated in patients with TIO and XLH, but not universally elevated in patients with ADHR [24], findings consistent with our work. Lorenz-Depiereux et al. [12] reported that FGF23 is at normal or low-normal serum levels in HHRH patients, which we also observed in our patient throughout the phosphate loading test. These findings show that the mechanism of hypophosphatemia in HHRH is independent of FGF23. Interestingly, as phosphate is a driver of FGF23, one might expect a transient increase in FGF23 in all groups after phosphate loading, but this was not seen. This adds to previous evidence showing that oral phosphate loading does not rapidly increase serum FGF23 levels in healthy men [25]. The patient's radiographic studies and prior surgeries reveal a longstanding history of severe nephrolithiasis. It has been reported that despite similar biochemical changes seen in heterozygous and homozygous carriers of SLC34A3 mutations, hypercalciuria does not lead to nephrolithiasis or nephrocalcinosis unless the affected individuals are inappropriately treated with vitamin D analogs [26,27]. Indeed, our patient had been treated with phosphate, calcium and 1,25(OH)2D supplementation for many years before the diagnosis of HHRH was established. Interestingly, some studies raise a possibility that some NaPi-IIc mutations such as g.4225_50del cause a dominant form of nephrolithiasis [16,17], but this hypothesis has not yet been proven. Further investigations are required to determine whether certain NaPi-IIc mutations are associated with renal calcification.
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An early and accurate diagnosis of HHRH has important therapeutic implications. Inorganic phosphate supplementation alone can cause a complete remission of HHRH, whereas the addition of vitamin D can create complications such as hypercalcemia, nephrocalcinosis, and renal damage. Nephrocalcinosis or nephrolithiasis can be the first symptoms of HHRH in patients who were mistreated with vitamin D and calcium, and therefore NaPi-IIc mutations should be considered in the differential diagnosis when these kidney findings are observed in the appropriate clinical context [17]. Hypercalciuria is frequent in the family members of HHRH patients. Thus, HHRH may be an underdiagnosed condition, mislabeled as idiopathic hypercalciuria or osteopenia. Furthermore, in Kremke et al.'s longitudinal follow-up study, they illustrated that vitamin D deficiency can mask some of the characteristic laboratory findings of HHRH. Thus, vitamin D levels should be checked and renal ultrasonography should be performed when considering the diagnosis of HHRH. Biochemical assessment and renal ultrasounds are also recommended for first-degree relatives of HHRH patients, especially when asymptomatic renal stones are present. There are some limitations in our study. We did not conduct a functional analysis of the mutant NaPi-IIc protein of our patient. Further studies are needed to determine the accurate function of NaPi-IIc in maintaining phosphate homeostasis. Conclusions Here we report the first case of HHRH in the Chinese population. The patient proved to be a compound heterozygote for a novel missense mutation and a previously described missense mutation of the gene SLC34A3. The discovery of this novel mutation adds to the list of more than 20 mutations for this disease. Results from an oral phosphate loading test further reveal that the mechanism of hypophosphatemia in HHRH is independent of FGF23. Disclosure page The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (No.81070687 and 81170805), National Science and Technology Pillar Program (2006BAI02B03), National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (Grant2008ZX09312-016), Beijing Natural Science Foundation (No. 7121012) and Scientific Research Foundation of Beijing Medical Development (No. 2007-3029). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2013.11.008. References [1] Tieder M, Modai D, Samuel R, Arie R, Halabe A, Bab I, et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985;312:611–7. [2] Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 2000;26:345–8.
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