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Annals of Clinical & Laboratory Science 39:182-187 (2009)
© 2009 Association of Clinical Scientists

Case Report

A Novel PHEX Mutation in a Korean Patient with Sporadic Hypophosphatemic Rickets

Juwon Kim1, Kyu Hyun Yang2, Ji Sun Nam3, Jong Rak Choi1, Jaewoo Song1, Myungsook Chang1 and Kyung-A Lee1
1 Departments of Laboratory Medicine, 2 Orthopaedic Surgery, and 3 Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

Address correspondence to Kyung-A Lee, M.D., Ph.D., Department of Laboratory Medicine, Yonsei University College of Medicine, 146-9 Dogokdong, Kangnamgu 146-92, Seoul, Korea; tel 82 2 2019 3531; fax 82 2 2019 4822; e-mail: KAL1119{at}yuhs.ac.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations including nonsense mutations, missense mutations, splicing-site mutations, insertions, and deletions in phosphate regulating genes on the X-chromosome (PHEX) are known to be responsible for X-linked hypophosphatemic rickets. The PHEX gene encodes an endopeptidase that is involved in phosphate regulation. Herein we present a female patient with sporadic hypophosphatemic rickets harboring a novel deletion mutation (c.1586_1586+1delAG; p.Glu529GlyfsX41) at exon 14 and intron 14 junction, which caused a premature termination at codon 569 and possibly produced a truncated PHEX protein. The laboratory and radiologic findings of the patient are reviewed to correlate the impact of the two-base deletion mutations at the exon-intron junction.

Keywords: phosphate regulating gene, PHEX mutations, X-chromosome, hypophosphatemic rickets


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Familial hypophosphatemic rickets (FHR) is defined as a group of disorders resulting from a defect in renal phosphate transport, which leads to phosphate wasting and hypophosphatemia. FHR is also characterized by abnormal regulation of vitamin D metabolism, resulting in normal 1,25-dihydroxyvitamin D concentrations despite hypophosphatemia [1]. The most common form of FHR is X-linked hypophosphatemic rickets (XLH) [2]. Autosomal dominant hypophosphatemic rickets (ADHR) [3] is known to be associated with mutations in FGF23 [4] and autosomal recessive hypophosphatemic rickets has been reported by different groups [5]. Patients with FHR features but without a family history of rickets, so called sporadic hypophosphatemic rickets, are common [6]. The gene that causes XLH was identified on Xp22.1, which was found to be a phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) [7], and experimental evidence showed that the recombinant PHEX functions as an endopeptidase [8]. The PHEX gene consists of 22 exons that translate into a 749 amino acid protein [9]. Recently, extensive mutation analysis of the PHEX gene showed a wide variety of gene defects including nonsense mutations, missense mutations, splicing site mutations, insertions, and deletions in different positions [10]. Only 14 mutations of the PHEX gene in Korean patients with hypophosphatemic rickets have been reported so far [11,12]. In this paper, we describe a novel deletion mutation at the splicing donor site of the PHEX gene, which is located at the exon-intron junction of exon 14 and the following intron (splice donor sequence). This mutation possibly caused a premature termination of the PHEX gene, which resulted in the severe phenotype of our patient.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
A 33-yr-old female of very short stature (140 cm, 45 kg) was admitted to the emergency room reporting leg pain. Physical examination revealed dental defects and deformities in the lower extremities including bowing of the legs. At the age of 2 yr, she had been referred to the Department of Pediatrics to evaluate walking dysfunction and growth retardation. She was diagnosed with rickets and started on medications. Her height at the age of 17 yr was 125.6 cm, and she had already undergone several surgical operations including corticotomies for multiple fractures. She had been regularly treated by a dentist for severe dental abscesses and had undergone multiple recurrent extractions. The patient had no family history of rickets and the rest of the family was healthy.

Laboratory analyses of serum revealed the following mean values (normal range): inorganic phosphate (P) 1.5 mg/ dl (2.1–5.6 mg/dl), calcium (Ca) 8.4 mg/dl (8.7–10.8 mg/dl), parathyroid hormone (PTH) 93.0 pg/ml (13–104 pg/ml), 25-hydroxyvitamin D3 8.7 ng/ml (7.6–75 ng/ml), 1,25-dihydroxyvitamin D3 38.0 pg/ml (20.1–46.2 pg/ml), osteocalcin 14.0 ng/ml (8.0–50.0 ng/ml), alkaline phosphatase 77 IU/L (42–117 IU/L), and creatinine (Cr) 0.5 mg/dl (0.5–1.4 mg/dl). The PTH was measured using an Immulite 2000 autoanalyzer (Diagnostic Products Corp.); 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and osteocalcin were measured on an E170 Analyzer (Roche Diagnostics Corp.). Serum concentrations of P and Ca were analyzed on a 7600 autoanalyzer (Hitachi).

Urine concentrations were: Ca 19.4 mg/dl, P 99.2 mg/dl, and N-telopeptide crosslink (NTx) 89.5 nM BCE/mM Cr (6.52–94.65 nM BCE/mM Cr). Urine concentrations of Ca and P were measured using a Hitachi 7180 autoanalyser and NTx was measured usinig the Vitros assay (Ortho Clinical Diagnostics) and creatinine levels in the urine. In order to estimate the renal capacity of phosphate reabsorption, maximum tubular capacity of phosphate (TmP) per unit volume of glomerular filtrate (TmP/GFR) was calculated using the Walton and Bijvoet nomogram [13]. The TmP/GFR of the patient was 1.2 mg/dl (2.5–4.5 mg/dl), which was low for her age.

Radiologic findings showed diffuse bony contour changes with osteoporotic changes in the distal femur and proximal tibia and fibulae due to underlying bony deformities and multiple previous fractures. Abnormal change in the pelvis was also noted (Fig. 1Go). Dual energy x-ray absorptiometry (DXA) (Hologic Discovery Co.) measurement showed mean bone mineral density (BMD) in the lumbar spine (L1-L4) and femoral neck of 1.325 g/cm2 and 0.601 g/cm2, giving T-scores of +2.8 and -2.2, respectively.


Figure 1
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  		Fig 1. X-rays of the lower limb show bowing of the tibia and fibulae with signs of multiple fractures at the intramedullary rod fixation. Increases in the bone diameter and cortical bone are evident with deformity of the pelvis.

 
For molecular analysis, after obtaining informed consent from the patient, genomic DNA was extracted from the peripheral blood leukocytes using an Easy DNA kit (Invitrogen) according to the manufacturer’s instructions. All 22 exons of the PHEX gene and the 3 exons of FGF23 and related exon-intron boundaries were individually amplified by PCR with the appropriate primers, as described previously [14], using a DNA thermal cycler (Perkin-Elmer Cetus). PCR products were then purified using a QIAquick Gel Extraction Kit (Qiagen) and directly sequenced using a cycle method with the same primers as the PCR and Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) in conjunction with an ABI Prism 310 automated genetic analyzer (Applied Biosystems). The sequence variations were described in relation to the cDNA reference sequence from a primary sequence database (GenBank, using Reference Sequence IDs NC_000023 [GenBank] and NM_000444 [GenBank] for PHEX, and NG_007087 [GenBank] and NM_020638 [GenBank] for FGF23; http://www.ncbi.nlm.nih.gov/Genbank/).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct nucleotide sequencing of the PHEX gene in leukocyte DNA from the patient revealed a novel 2 base deletion mutation (c.1586_1586+1delAG; p.Glu529GlyfsX41) in exon 14 and intron 14 junction that changed the Glu at codon 569 to a stop codon, which suggests a possible production of truncated PHEX molecules (Fig. 2Go). There was no mutation found in the FGF23 gene.


Figure 2
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  		Fig 2. Mutational analysis of exon 14 of the PHEX gene shows a 1-bp deletion of exon 14 and a 1-bp deletion of the first nucleotide of intron 14, which is the splice donor sequence.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found a novel 2 base pair deletion mutation (c.1586_1586+1delAG; p.Glu529GlyfsX41) in the exon 14 and intron 14 junction that caused a premature termination at codon 569, which presumably produced a truncated PHEX protein. The laboratory and radiologic findings of the patient were reviewed to correlate the impact of the deletion mutation in the exon-intron junction. The mutation lies within the splice donor sequence of exon 14 and intron 14 according to the PHEX gene structure information documented by Francis et al [9]. Only 14 mutations have been identified in Korean patients [11,12] with XLH, and this is the first splicing-site mutation reported on exon 14 of the PHEX gene in a Korean patient. In addition, this was a sporadic case and the patient did not have a familial history of this mutation. There were comparatively more sporadic cases (71%) in a study conducted in Korea compared with familial forms accounting for 77% and 86% of patients involved in the studies conducted by Dixon et al [24] and Francis et al [9], respectively. This supports the idea that there might be a higher incidence of de novo occurrence of the PHEX mutation in the Korean population [12]. Since there is evidence of sporadic cases subsequently transmitting the phenotype in an X-linked dominant manner consistent with XLH [6], it is also meaningful to screen for such mutations in newly diagnosed patients and their family members in order to provide appropriate genetic counseling.

It is important to obtain information of the genotype-phenotype relationship, which enables us to predict the severity and outcome produced by the mutation since several groups have reported that there seems to be no apparent hot spots for this mutation. Holm et al [15] reported that the gene structure and exon-intron borders are conserved between PHEX, neutral endopeptidase (NEP), and endothelin-converting enzyme-1 (ECE-1) and deletion in the splice donor site is expected to result in exon skipping. This suggests that the exon-intron border is crucial in the formation of functional PHEX proteins and the mutation in this region probably contributes to the severe phenotypes seen in the patients with XLH. In our patient, deletion mutation at the exon-intron border caused a premature stop which subsequently caused severe clinical symptoms including recurrent fractures in the lower extremities. She had undergone more than 7 osteotomies and had evident dental defects including formation of abscesses and recurrent extractions. Severe bowing of the leg and her short stature were prominent as well. It was reported that BMD in the lumbar spine is increased while BMD in the radial diaphysis, primarily consisting of cortical bone, is decreased [16,17] in patients with phosphatemic rickets (as was the case with our patient). The specific reason for this effect still needs to be elucidated.

To date, 197 different PHEX gene mutations have been reported in XLH patients [18] and only 6 mutations including 1 deletion, 3 frameshifts, 1 missense, and 1 splice-site mutation have been found in exon 14. Although the correlation between genotype and phenotype is still under investigation, many reports have proved that the mutation causing premature stops that result in the production of truncated proteins is certainly associated with more severe clinical consequences. Furthermore, mutations involving the splice site seem to show a more severe clinical picture. Interestingly, Holm et al [1] reported a case harboring c.1584+3delGAGT mutation, which was a 4 bp deletion mutation in the same location of our case [1]. The severity of the disease was the same as or more severe in our case, which harbored a 2 bp deletion right on the exonintron junction. It was hypothesized that a mutation involving the 2 bases on the exon-intron junction are sufficient to interfere with the splicing mechanism. Therefore, no matter how many bases are deleted in addition to the exon-intron junction bases, the effect would be similar in terms of clinical severity. Thirty-nine splice-site mutations reported in the PHEX gene are summarized in Table 1Go. Of 29 subjects whose mode of inheritance was documented, 19 were X-linked and 10 were sporadic. No significant correlations between severity of the disease and location of the mutation were observed.


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  		Table 1. Summary of splice site mutations found in the PHEX gene

 
Since sporadic diseases can be caused by other types of hypophosphatemic rickets, such as ADHR, a mutational study on FGF23 was carried out concomitantly with the PHEX mutational study, and no mutation was found on FGF23. Recent studies have shown that the increased circulating levels of FGF23 was associated with PHEX mutation in XLH patients [19], and suggested that overexpression of FGF23 is probably the ultimate link in the pathogenesis of XLH [20]. Unfortunately, the serum level of FGF23 could not be determined due to lack of the patient’s serum sample.

In conclusion, we present a female patient with XLH harboring a novel deletion mutation in the exon-intron junction of the PHEX gene, which might have caused a severe clinical phenotype in our patient. Recently, treatment has changed depending on the different genetic defects, so that disorders caused by low 1,25(OH)2D levels due to FGF23 are recommended to be treated with oral phosphate or 1,25(OH)2D3, whereas hypophosphatemia due to the mutations in sodium phosphate co-transporters named NPT2a or NPTc should be treated with oral phosphate alone [30]. Thus, gathering information on a case-by-case basis is valuable in elucidating the molecular pathogenesis of hypophosphatemic rickets and may aid in developing new and better treatment strategies.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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