DOI 10.1515/jpem-2013-0270      J Pediatr Endocr Met 2014; 27(7-8): 787–789

Patient report Devi Dayal*, Sheetal Sharda, Savita Verma Attri and Rakesh Kumar

Hypophosphatemic rickets caused by a novel PHEX gene mutation in an Indian girl Abstract: We report a girl who presented with clinical and biochemical features of hypophosphatemic rickets. Mutational analysis detected a heterozygous nonsynonymous sequence variation in exon 11 of the PHEX gene (NM_000444.4:c.1216T > C, NP_000435.3:p.Cys406Arg). This previously undescribed PHEX mutation is probably the cause of renal phosphate wasting in our patient that resulted in rickets. Keywords: children; hypophosphatemic rickets; Indian ancestry; novel mutation; PHEX gene; X-linked hypophosphatemia. *Corresponding author: Devi Dayal, Department of Pediatrics, Advanced Pediatrics Center, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India, Phone: 0091-172-2755657, Fax: 0091-172-2744401/2745078, E-mail: [email protected] Sheetal Sharda, Savita Verma Attri and Rakesh Kumar: Department of Pediatrics, Advanced Pediatrics Center, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India

Introduction Of all the heritable forms of hypophosphatemic rickets, X-linked hypophosphatemia (XLH) is the most common affecting about 1 in 20,000 newborns (1). The phenotypic spectrum of XLH ranges from isolated hypophosphatemia to severe lower extremity bowing. In children, this X-linked dominantly inherited disorder is characterized clinically by rickets, disproportionate short stature, lower extremity deformities, bone pain, poor dental development and tooth abscesses (1). Laboratory features include low serum phosphate, normal serum calcium, normal or increased parathyroid hormone (PTH), and inappropriately normal or decreased concentrations of 1,25-dihydroxyvitamin D (1,25 OHD). In addition, phosphaturia can be demonstrated. Rachitic changes and decreased bone density often are marked more in the peripheral as compared to the axial skeleton (2).

The genetic basis of XLH is inactivating mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX ) that lead to increased bone expression of the fibroblast growth factor 23 (FGF23), which, through its action on proximal renal tubules, decreases renal tubular phosphate reabsorption, resulting in hypophosphatemia (1, 3). Up to this time, 329 different mutations in the PHEX gene have been described in patients with hypophosphatemic rickets and include nonsense mutations, missense mutations, splice (acceptor/donor) site mutations, frameshift mutations, insertions and deletions at different positions (4). The majority of these mutations are de novo and occur sporadically. The female dominance (female/male ratio of 5:1) in sporadic cases is observed in all ethnic backgrounds (5). Although XLH commonly is reported from the Indian subcontinent, patient reports with documented PHEX mutations are rare (6). In this brief communication, we present the case of a 4-year-old girl who had major clinical and laboratory features of hypophosphatemic rickets and was detected to have a novel missense mutation within the PHEX gene.

Case report A 4-year-old girl presented with history of long standing rickets that had failed to respond to usual treatment with vitamin D and calcium supplements. Her parents noticed deformity of lower limbs for about 1 year, which progressed slowly. There was no history to suggest any systemic illness, dietary deprivation, or fractures. She was born to nonconsanguineous parents at term and had a normal birth weight and length. There was no family history of any bone disease. Her parents and an elder brother were asymptomatic. On examination her weight was 12.6 kg (–2.0 SDS on WHO growth charts 2006) and height 92.5 cm (–2.2 SDS). She had anterolateral bowing of thighs and genu varum deformity of lower limbs. Her dentition was normal for age. Systemic examination was unremarkable.

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788      Dayal et al.: Hypophosphatemic rickets caused by a novel PHEX gene mutation in an Indian girl Radiographic examination of long bones showed metaphyseal cupping and fraying and generalized osteopenia. Dual energy X-ray absorptiometry (DXA) measurements showed a severely reduced mean bone mineral density (BMD) in the lumbar spine (L2–L4) of 0.525 g/cm2 representing a T-score of –4.40. Laboratory investigations revealed normal levels of hematological parameters and blood urea, serum creatinine, lactate, and electrolytes. Serum calcium was 9.2 mg/dL, phosphorus 2.6 mg/dL (normal 3.7–5.6 mg/dL) and alkaline phosphatase (ALP) 776 U/L (normal 145–420 U/L). Parathyroid hormone (PTH) level was 5.2 pmol/L (normal 0.95–6.8 pmol/L). Plasma level of 25-hydroxyvitamin D was 62.87 nmol/L (normal, 50–250 nmol/L) and 1,25 OHD was 56 nmol/L (normal 60–108 nmol/L). Spot urine calcium creatinine ratio was 0.12. Urine concentrations of phosphorus and creatinine were 21.1 mg/dL and 51.2 mg/dL, respectively. The calculated maximum tubular capacity of phosphate (TmP) per unit volume of glomerular filtrate (TmP/GFR) was 0.93 mmol/L (normal, 1.15–2.44 mmol/L) (7). Genomic DNA of the patient and her unaffected mother were extracted from whole blood samples collected in ethylenediaminetetraacetic acid (EDTA) tubes using Qiagen whole blood DNA extraction kit (Qiagen Corporation, Valencia, CA, USA). All 22 exons and the region around the presumable polyadenylation signal of the PHEX gene were amplified with intronic primers using polymerase chain reaction (PCR). The resulting PCR products were sequenced directly on both the sense and antisense strand using an automated ABI PRISM 3730 sequencer (Life Technologies, Foster City, CA, USA). We found a nonsynonymous sequence variation (nsSNV) within exon 11 of the PHEX gene (NM_000444.4:c.1216T > C, NP_000435.3:p.Cys406Arg) in the DNA of the affected girl in a heterozygous state (Figure 1). This sequence variation was confirmed in NM_000444.4:c.1216T>C, NP_000435.3:p.Cys406Arg Gln

Trp

Asp

Lys

Cys/Arg Val Asn

Phe Iso

Glu

Patient

Gln

Trp

Asp

Lys

Cys

Val Asn

Phe Iso

Glu Mother

Figure 1 Mutation analysis of the PHEX gene in the affected girl and her mother. The mutation is indicated above the electropherograms. The affected patient is heterozygous for a nonsynonymous sequence variation in exon 11 of the PHEX gene.

a second independent PCR and sequence reaction. The nonsynonymous sequence variation was not found in the DNA of the mother (Figure 1). A DNA sample of the father was not available. The child was started on calcitriol 0.25 μg/day and elemental phosphorus 1 g/day in five divided dosages. She has been on regular followup with us for the past 2 years and has not shown any progression of limb deformities. Her ALP and phosphorus levels have remained normal for age.

Discussion Our patient exihibited typical clinical features characteristic of hypophosphatemic rickets. The low TmP/GFR in presence of a low serum phosphate is very characteristic of XLH. Because of lack of availability, we could not measure FGF23 levels in serum or plasma, which usually are elevated in almost all patients with XLH and which may be an important indicator to diagnose XLH (8). Urinary calcium creatinine ratio of 0.12 excluded the possibility of hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in this patient. To date, the nonsynonymous sequence variation (nsSNV) identified in our patient is not described in the literature, the database of Human Gene Mutation (HGMD, Cardiff, UK), the PHEX-specific database (www.phexdb. mcgill.ca) (4), the database of short genetic variations (dbSNP 137, NCBI), or in more than 2500 mostly Caucasian in-house control individuals. Hence we considered the identified nsSNV as a new mutation. In a previous study in a Chinese population, another mutation (c.1216T > A, p.Cys406Ser) at the same position as ours is described in one of the patients (9). The T > A change in that patient is a transversion, whereas the T > C change in our patient is a transition. Moreover, the result is an amino acid change from cystein to arginine (positively charged side chain) in our case and no charge change in the Chinese case (cystein to serine). Hence, the mutation in our patient is clearly different from the previously described case. The identified nsSNV was predicted to be probably damaging using PolyPhen as prediction tool for functional effects of human nsSNVs (http://genetics.bwh.harvard. edu/pph/). The cystein at position 406 is highly conserved in other species and in the neutral endopeptidase protein family, suggesting an important role in the PHEX protein structure or function. Mutations at this position likely would result in protein structure change and render the PHEX protein nonfunctional. Our patient’s phenotype also

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Dayal et al.: Hypophosphatemic rickets caused by a novel PHEX gene mutation in an Indian girl      789

was consistent with the loss of PHEX function, probably as a result of this mutation. The mutation in our patient appears to be de novo or paternally derived. It has been suggested that de novo X-linked mutations may occur mainly in paternal derived X chromosomes, thus affecting only females (10). Male mutation bias is common as males undergo many more germ line cell divisions than females and accumulate more DNA replication errors in their germ line (10). As most sporadic patients are female, it is likely that mutated PHEX alleles resulted from mutagenesis in X chromosomes of paternal germ cells and PHEX mutagenesis in paternal germ cells occurs frequently in sporadic patients (11).

In conclusion, XLH in our patient is caused by a novel mutation within exon 11 of the PHEX gene. This appears to be the second report of a new PHEX mutation in a patient of Indian ancestry. Acknowledgments: We thank Bettina Lorenz-Depiereux and Tim Strom, Institute of Human Genetics, Klinikum rechts der Isar, Technische Universität, München, Germany for molecular diagnostics of the PHEX gene and Sandy Lösecke for excellent technical assistance. Received June 26, 2013; accepted February 28, 2014; previously published online April 16, 2014

References 1. Roth KS, Ward RJ, Chan JC, Sarafoglou K. Disorders of calcium, phosphate and bone metabolism. In: Sarafoglou K, Hoffmann GF, Roth KS, editors. Pediatric endocrinology and inborn errors of metabolism. New York: McGraw Hill, 2009:619–64. 2. Shore RM, Langman CB, Poznanski AK. Lumbar and radial bone mineral density in children and adolescents with X-linked hypophosphatemia: evaluation with dual X-ray absorptiometry. Skeletal Radiol 2000;29:90–3. 3. Francis F, Henning S, Korn B, Reinhardt R, de Jong P, et al. A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nat Genet 1995;11:130–6. 4. Sabbagh Y, Jones AO, Tenenhouse HS. PHEXdb, a locus-specific database for mutations causing X-linked hypophosphatemia. Hum Mutat 2000;16:1–6. 5. Durmaz E, Zou M, Al-Rijjal RA, Baitei EY, Hammami S, et al. Novel and de novo PHEX mutations in patients with hypophosphatemic rickets. Bone 2013;52:286–91. 6. Chandran M, Chng CL, Zhao Y, Bee YM, Phua LY, et al. Novel PHEX gene mutation associated with X linked

hypophosphatemic rickets. Nephron Physiol 2010; 116:17–21. 7. Payne RB. Renal tubular reabsorption of phosphate (TmP/GFR): indications and interpretation. Ann Clin Biochem 1998;35:201–6. 8. Igaki JM, Yamada M, Yamazaki Y, Koto S, Izawa M, et al. High iFGF23 level despite hypophosphatemia is one of the clinical indicators to make diagnosis of XLH. Endocr J 2011;58:647–55. 9. Yue S. Hypophosphatemic rickets/osteomalacia genetics research fibrous dysplasia of bone resistance gene mutation detection. China Union Medical Information, Internal Medicine 2010. http://www.dissertationtopic.net/doc/1539800 . Accessed 20 December, 2013. 10. Zhu X, Li M, Pan H, Bao X, Zhang J, et al. Analysis of the parental origin of de novo MECP2 mutations and X chromosome inactivation in 24 sporadic patients with Rett syndrome in China. J Child Neurol 2010;25:842–8. 11. Goetting-Minesky MP, Makova KD. Mammalian male mutation bias: impacts of generation time and regional variation in substitution rates. J Mol Evol 2006;63:537–44.

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Hypophosphatemic rickets caused by a novel PHEX gene mutation in an Indian girl.

We report a girl who presented with clinical and biochemical features of hypophosphatemic rickets. Mutational analysis detected a heterozygous nonsyno...
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