CLINICAL REPORT
Unusually Severe Hypophosphatemic Rickets Caused by a Novel and Complex Re-Arrangement of the PHEX Gene Tuula Pekkarinen,1* Bettina Lorenz-Depiereux,2 Martina Lohman,3 and Outi Ma¨kitie4,5,6 1
Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, Vantaa, Finland
2
Institute of Human Genetics, Helmholtz Center Munich, Neuherberg, Germany HUS Medical Imaging Center, Helsinki University Hospital, Helsinki, Finland
3 4
Pediatric Endocrinology and Metabolic Bone Diseases, Children’s Hospital, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland 5 Folkha¨lsan Institute of Genetics, Helsinki, Finland 6
Department of Molecular Medicine and Surgery, Karolinska Institutet, and Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden Manuscript Received: 24 March 2014; Manuscript Accepted: 9 July 2014
X-linked hypophosphatemia (XLH) is caused by mutations in PHEX. Several other genetic forms of hypophosphatemia have also been described. These disorders share variable clinical presentation ranging from mild hypophosphatemia to severe lower extremity bowing. We report on a 43-year-old woman with short stature, painful leg deformities, and poor dentation. Her biochemical profile showed hypophosphatemia with renal phosphate wasting. Due to unusually severe clinical presentation and absence of mutations in Sanger sequencing of the PHEX gene, quantitative multiplex ligation-dependent probe amplification was performed. A large deletion within the PHEX gene encompassing exons 8 to 11 was identified. We generated a specific junction fragment using long-range PCR and sequenced the junction fragment to determine the exact deletion breakpoints. We found a heterozygous novel complex re-arrangement involving gross deletions, insertions, and inversion of PHEX (hg19: g.22,115,003_22,141,395del;g:22,145,536_22,150,789delinsCins22, 114,640_22,114,698invinsA). Thus, the complex re-arrangement including a deletion of coding exons 8 to 11 of the PHEX can be regarded as the cause of XLH in the patient reported here. Phosphate and active vitamin D treatment was initiated with subsequent relief in bone pain and physical improvement. This report expands the spectrum of clinical severity underlying genetic defects in XLH and highlights the importance of conventional medical therapy even at adult age. Furthermore, our findings underscore the importance of search for gene deletions in patients with suspected XLH. Ó 2014 Wiley Periodicals, Inc.
Key words: X-linked hypophosphatemia; novel deletions/ insertion in PHEX gene; severe phenotype
INTRODUCTION Hypophosphatemia is an established cause of rickets. The most common inherited form of hypophosphatemic rickets is X-linked
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Pekkarinen T, Lorenz-Depiereux B, Lohman M, Ma¨kitie O. 2014. Unusually severe hypophosphatemic rickets caused by a novel and complex re-arrangement of the PHEX gene. Am J Med Genet Part A 164A:2931–2937.
hypophosphatemia (XLH; OMIM 307800), which results from mutations in the PHEX (phosphate regulating endopeptidase homolog, X-linked) and is inherited in an X-linked dominant pattern [Anonymous, 1995; Carpenter et al., 2011]. A few other genetic forms of hypophosphatemia have also been described. They differ from XLH by their inheritance pattern but clinical course, biochemistry and radiographic features overlap. XLH may result from reduced cleavage and consequently increased concentration of the phosphaturic protein fibroblast growth factor 23 (FGF23), which is made by osteocytes and activates renal KLOTHO/FGFR1 receptor heterodimers to inhibit renal phosphate reabsorption and Conflict of interest: none Grant sponsor: Folkha¨lsan Research Foundation; Grant sponsor: Sigrid Juselius Foundation; Grant sponsor: Academy of Finland; Grant sponsor: Swedish Research Council. Correspondence to: Tuula Pekkarinen, Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, Sairaalakatu 1, Vantaa, P.O.Box 900, FI-00029 HUS. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 13 August 2014 DOI 10.1002/ajmg.a.36721
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1,25-dihydroxyvitamin D synthesis [Strom and Juppner, 2008]. Gain-of-function mutations in the FGF23 may render it resistant to cleavage and underlie the autosomal dominant form of hypophosphatemic rickets (ADHR, MIM 193100) [ADHR Consortium, 2000]. In addition, two autosomal recessive forms of hypophosphatemic rickets (ARHR1 OMIM 241520, ARHR2 OMIM 613312) have recently been described. These are caused by inactivating mutations in the DMP1 (dentin matrix protein) and ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) respectively, encoding proteins also involved in the regulation of FGF23 [Feng et al., 2006; Lorenz-Depiereux et al., 2006; LorenzDepiereux et al., 2010; Levy-Litan et al., 2010]. The diagnosis of XLH can be challenging. It should be considered if bowing of legs and delayed growth are present in early childhood [Stickler et al., 1970; Holm et al., 2001], but it may be misdiagnosed as nutritional rickets, metaphyseal dysplasia or physiological bowing [Carpenter et al., 2011]. However, early diagnosis is important as treatment during growth partially corrects leg deformities and improves adult height [Ma¨kitie et al., 2003; Quinlan et al., 2012]. Autosomal dominant and recessive hypophosphatemic rickets share common clinical features with XLH but are far rarer and show different inheritance pattern; therefore genetic analysis has been proposed to be mandatory for proper diagnosis of hypophosphatemias [Saito et al., 2009]. During the last two decades, increased knowledge of the various forms of hypophosphatemia has improved diagnostic accuracy and enabled development of treatment guidelines [Carpenter et al., 2011]. We report here on a woman with unusually severe clinically diagnosed hypophosphatemic rickets, which was genetically confirmed as XLH at the age of 43.
CLINICAL REPORT This study was approved by the Research Ethics Board of the Helsinki University Central Hospital, and the patient gave her written informed consent to participation. Because of severe bone pain and leg deformities, this 43-year-old woman of Middle East origin was referred to the Division of Endocrinology, Helsinki University Central Hospital, after her immigration to Finland in 2010. No previous medical records were available, but she described progressive lower limb bowing after age of two. In early childhood, she was treated with intramuscular vitamin D injections in her native country. After age 6, she underwent numerous orthopedic procedures, like osteotomies, followed by immobilization in plaster. Her physical impairment progressed and growth decelerated after these unsuccessful operations. Her family members were of normal height and healthy, and there was no history of parental consanguinity. She complained of bone pain mainly in the legs. She had difficulties to walk and used crutches. She required substantial personal help and assistive equipment to manage everyday life. On physical examination, her height was 127 cm and body weight 47.7 kg. She had four permanent teeth left and used dental prosthesis. Hearing was normal. Right leg was severely deformed, possibly due to previous operations, and amputation was considered. She had coxa vara but knee and hip movements were normal; elbow extension was limited. Laboratory evaluation showed moderate hypophosphatemia and renal phosphate wasting with normal parathyroid hormone level, no hypercalciuria, vitamin D deficiency, and normal renal function (Table I). There was no sign of anemia, coeliac disease or
TABLE I.. Biochemical and Bone Density Findings of the Patient Parameter Ca-ion (mmol/l/pH7.4) Phosphorus (mmol/l) Magnesium (mmol/l) 25(OH) vitamin D (nmol/l) 1,25(OH)2vitamin D (pmol/l) Alkaline phosphatase (U/l) Parathyroid hormone (ng/L) FGF-23 (ng/L) P-Creatinine (mmol/l) P-Albumin (g/l) 24 h U-Ca (mmol/24 h) 24 h U-Pi (mmol/24 h) 24 h U volume (l/24 h) Tubular reabsorption of phosphorusa (mmol/L) U-INTP (nmol/mmol Creatinine) PINP (mg/L) Lumbar spine BMD Z-scorec Femoral neck BMD Z-score right/leftc a
1 year on therapy 1.19 0.54 – 40 100 113 69b – 38 0.65 54.0 2.1 – 40 46.8 – –
Based on 2-hour samples, calculated by use of the equation: serum Pi-(Urine Pi x serum creatinine/urine creatinine). Reference range 12–47 ng/l. Measured by dual-energy X-ray absorptiometry using A Lunar Prodigy scanner (Lunar Radiation Corp., Madison, WI, USA.).
b c
Before therapy 1.15 0.49 0.74 41 124 82 67 62 37 36.0 0.91 17.7 1.1 0.45 67 32.4 þ 2.7 1.9/1.0
2 years on therapy 1.20 0.69 – 52 – – 44b – 35 – 1.01 60.2 1.6 – – – – –
Reference range 1.16 – 1.3 0.76 – 1.41 0.71 – 0.94 > 40 45 – 165 35 – 105 8 – 73 10 – 50 50 – 90 36 – 45 1.3 – 6.5 20 – 50 – – < 65 19 – 84 – –
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Fanconi syndrome, no glucosuria or proteinuria. At the time of referral, she was using daily calcium supplementation (500 mg) with cholecalciferol (10 mg) and etoricoxib for pain. Radiographs showed non-homogeneous bone structure and coarse trabeculation of the metaphyseal regions. The long bones of the lower limbs were short, abnormally shaped and severely bowed. Pseudofractures and enthesopathy were seen (Fig. 1). Periosteal calcification was seen in forearms (Fig. 2). Several teeth were lacking but no hyperostosis was seen in the skull (Fig. 3). The lower lumbar spine showed narrowing of the central spinal canal (Fig. 4). Oral phosphate supplement was commenced with 2250 mg (50 mg/kg) daily divided into four doses; this was combined with vitamin D analogue alfacalcidiol 0.5 mg twice daily and
FIG. 1. Standing weight bearing full-length radiographs of the lower extremities showing severe bowing of bilateral femurs and right tibia. Several insufficiency fractures (Looser’s zones) are seen (arrows). Entesopathy is seen at the insertion areas of Musculus rectus femoris bilaterally ( ). Signs of arthritis of the hips with sclerosis and limited intra-articular spacing are seen.
FIG. 2. A-P-radiograph of the left lower arm shows ossification along the cortexes of the radius diaphysis (arrow).
cholecalciferol 20 mg, later 40 mg daily. Her bone pain diminished within weeks after treatment onset and gait improved; she was able to walk with one elbow crutch and short distances even without assistance. Table I shows laboratory data during treatment. Later,
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FIG. 3. Lateral radiograph of the skull shows lacking upper teeth and only a few lower teeth, but no hyperostosis of the skull.
orthopedic operations to correct lower limb deformities were started. Due to the observed clinical symptoms, we decided to analyze PHEX by capillary sequencing. DNA was extracted from peripheral blood. All the 22 exons and the region around the presumable polyadenylation signal of the PHEX were amplified with intronic primers using PCR and sequenced on both the sense and antisense strand. No mutations within the entire coding region and the splice junctions of the PHEX were identified by Sanger sequencing. As 20– 30% of PHEX mutations are gross deletions [Morey et al., 2011], which span one or more exons and which cannot be detected by PCR and sequencing in female patients, we decided to use a quantitative approach (multiplex ligation-dependent probe amplification MLPA, MRC Holland) to search for dosage differences within the 22 exons. We found a heterozygous deletion of PHEX exons 8–11. We investigated this further by performing long-range PCR with primers in intron 7 and 12 of the PHEX gene to determine the deletion breakpoints. We generated a long-range PCR product of approximately 7 kb, and subsequently performed direct sequencing to identify the deletion breakpoints. Upon identification of the breakpoints, we subjected our findings to BLAT (UCSC Browser), and finally found a complex re-arrangement consisting of a large deletion of 27.7 kb (hg19:g.[22,115,003_22,141,395del(;) ¼ ]; NM_000444.5:c.[850-433_1302 þ 18085del(;) ¼ ], NP_000435.3: p.[Ile284_Met434del(;) ¼ ]) including exons 8–11 of the PHEX coupled by a second smaller proximal deletion/insertion of 5.2 kb. We found a duplication of 58 bp of intron 7 (hg19: g.22,114,640_22,114,698) inverted and inserted within the smaller deletion including two single basepair appendices at the 50 (C) and 30 (A) end of the inversion (hg19:g:22,145,536_22,150,789delins-
FIG. 4. Lateral radiograph of the lumbar spine shows narrowing of the spinal canal in the lower lumbar spine (arrows) due to short pedicles, enlarged facet joints, and spondylosis.
Cins22,114,640_22,114,698invinsA) (Fig. 5). In addition, we were not able to amplify the normal full-length allele of 38.6 kb by long-range PCR.
DISCUSSION We describe a middle-aged hypophosphatemic woman in whom a novel complex re-arrangement of the PHEX was identified. Her phenotype includes poor dentation, short stature, childhood-onset severe lower limb varus deformity requiring osteotomies and causing severe physical impairment with pain in adulthood. Her early treatment, vitamin D injections, was the treatment of choice in XLH 40 years ago, suggesting that she was diagnosed in childhood but did not have access to conventional phosphate and active vitamin D therapy prior to immigration. Reports concerning morbidity of untreated adult patients with a clinical diagnosis of XLH have revealed that symptomatic
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FIG. 5. Location of the complex re-arrangement involving gross deletions, insertions, and inversion of the PHE X gene in the patient.
osteomalacia, enthesopathy, joint pain, dental problems and degenerative joint disease arising from lower limb deformities are common [Reid et al., 1989]. Typical radiographic features in the appendicular skeleton of vitamin D treated adults include abnormally bowed long bones, looser zones due to insufficiency fractures, osteoarthritis in ankles, knees, feet, sacroiliac joints and wrists, abnormally angular (trapezoidal) distal femoral condyles, shortening of talar neck and flattening of the talar dome and accessory ossicles. Other common findings are spinal stenosis and enthesopathy [Hardy et al., 1989]. Our patient demonstrated several of these radiographic findings. A wide variation in the severity and clinical manifestations of untreated XLH patients, even within the same family is typical [Stickler et al., 1970]. Recently, Beck-Nielsen et al. [2010] identified through family screening genetically verified, untreated XLH adults who displayed very few symptoms. Compared with these reports, the presentation of our patient seemed to be unusually severe. Since the 1980’s, childhood-onset treatment with biologically active vitamin D and phosphate has greatly improved bone mineralization and growth of patients with XLH. In adults, medical therapy is empirical and the goal is clinical improvement. Candidates for treatment are adult patients with spontaneous insufficiency fractures, biochemical evidence of osteomalacia and disabling skeletal pain, and those pending orthopedic procedures [Carpenter et al., 2011]. Therapy should start six months prior to the intervention and continue until bone healing is complete. In our patient, treatment initiation at the age of 43 resulted in rapid pain relief and gait improvement, and subsequent orthopedic surgeries were successful. Low serum concentrations of phosphate and 1,25(OH)2 vitamin D and decreased renal phosphate reabsorption observed here are characteristic for XLH. Normal serum alkaline phosphatase was surprising, but it has been reported among adult XLH patients regardless of treatment [Ruppe, 2012]. High serum FGF23 despite hypophosphatemia is one indicator of XLH. Here the FGF23 level was only mildly elevated. Igaki et al. [2011] did not find any correlation between circulating FGF23 levels and measures of
disease severity in XLH, suggesting that the link between XLH severity and serum FGF23 is probably not straightforward. Despite the fact that XLH is a mineralization defect with osteomalacia, elevated z-score of lumbar spine areal bone mineral density was observed in our patient and has been reported previously [Reid et al., 1989; Rosenthall, 1993; Beck-Nielsen et al., 2010; Cheung et al., 2013]. Among adults, extra-osseous calcifications or secondary degenerative findings in the lumbar spine may explain this [Hayirlioglu et al., 2009; Carpenter et al., 2011]. PHEX is a relatively large gene with 22 coding exons. Several mutations have been described, with no mutational hotspots. Studies evaluating genotype–phenotype correlations in XLH have suggested a correlation between more severe bone disease, as determined by the severity of deformity and a history of osteotomies, and truncating mutations [Holm et al., 2001] or mutations in the C-terminal region [Song et al., 2007]. Another study showed that clearly deleterious PHEX mutations (nonsense mutations, insertions or deletions, and splice site mutations leading to premature stop codons) associated with lower tubular phosphate reabsorption and lower calcitriol levels when compared to plausibly deleterious mutations (missense changes or in-frame deletions) [Morey et al., 2011]. Our patient had a previously undescribed complex re-arrangement including a large deletion involving exons 8–11 of the PHEX, and therefore this change is likely to significantly alter the function of the PHEX protein. This report highlights the importance of careful genetic analysis of PHEX with not only normal sequence analysis but with multiplex ligation-dependent probe amplification (MLPA) or comparable methods to detect deletions and/or duplications. Various studies have shown that direct sequencing of the coding regions and intronic borders of PHEX detect mutations in 57–78% of individuals with the clinical and biochemical diagnosis of XLH [Holm et al., 1997; Dixon et al., 1998; Ichikawa et al., 2008; Gaucher et al., 2009; Ruppe et al., 2011]. When both exon sequencing and MLPA analysis are used, mutations can be detected in a significantly larger proportion of patients [Clausmeyer et al., 2009; Morey et al., 2011; Beck-Nielsen et al., 2012].
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CONCLUSION Management of severe bone deformities of XLH in adulthood is highly complex and challenging. Therefore, with this case we highlight the significant clinical variability in XLH, which is further impacted by the timing of medical therapy and success of surgical interventions. Nowadays, early diagnosis enables early initiation of proper treatment which in turn improves height outcome and reduces the need for surgical interventions. The care of XHL patients should be centralized to specialist referral centers. A genetic diagnosis enables appropriate genetic counseling and prenatal or preimplantation diagnostics and should nowadays be regarded a part of diagnostic work-up. In XLH, deletions in the PHEX are frequently observed, and these should be sought when Sanger sequencing fails to detect a causative mutation.
ACKNOWLEDGMENTS We thank Tim Strom for his contribution to this work and Sandy Lo¨secke for excellent assistance. Financial support was provided by grants from Folkha¨lsan Research Foundation, Sigrid Juselius Foundation, the Academy of Finland and the Swedish Research Council.
REFERENCES A gene (PEX) with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. 1995. The HYP Consortium Nat Genet 11:130–136. ADHR Consortium. 2000. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet 26:345–348. Beck-Nielsen SS, Brusgaard K, Rasmussen LM, Brixen K, Brock-Jacobsen B, Poulsen MR, Vestergaard P, Ralston SH, Albagha OME, Poulsen S, Haubek D, Gjorup H, Hintze H, Andersen MG, Heickendorff L, Hjelmborg J, Gram J. 2010. Phenotype presentation of hypophosphatemic rickets in adults. Calcif Tissue Int 87:108–119. Beck-Nielsen SS, Brixen K, Gram J, Brusgaard K. 2012. Mutational analysis of PHEX, FGF23, DMP1, SLC34A3 and CLCN5 in patients with hypophosphatemic rickets. J Hum Genet 57:453–458. Carpenter TO, Imel EA, Holm IA, Jan de Beur SM, Insogna KL. 2011. A clinician’s guide to X-linked hypophosphatemia. J Bone Miner Res 7:1381–1388. Cheung M, Roschger P, Klaushofer K, Veilleux LN, Roughley P, Glorieux FH, Rauch F. 2013. Cortical and trabecular bone density in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 98:E954–E961. Clausmeyer S, Hesse V, Clemens PC, Engelbach M, Kreuzer M, BeckerRose P, Spital H, Schulze E, Raue F. 2009. Mutational analysis of the PHEX gene: Novel point mutations and detection of large deletions by MLPA in patients with X-linked hypophosphatemic rickets. Calcif Tissue Int 85:211–220. Dixon PH, Christie PT, Wooding C, Trump D, Grieff M, Holm I, Gertner JM, Schmidtke J, Shah B, Shaw N, Smith C, Tau C, Schlessinger D, Whyte MP, Thakker RV. 1998. Mutational analysis of PHEX gene in X-linked hypophosphatemia. J Clin Endocrinol Metab 83:3615–3623. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, Zhang S, Drezler MK, Quarles LD, Bonewald LF, White KE. 2006. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38:1310–1315. Gaucher C, Walrant-Debray O, Nguyen TM, Esterle L, Garabe´dian M, Jehan F. 2009. PHEX analysis in 118 pedigrees reveals new genetic clues in hypophosphatemic rickets. Hum Genet 125:401–411.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Hardy DC, Murphy WA, Siegel BA, Reid IR, Whyte MP. 1989. X-linked hypophosphatemia in adults: Prevalence of skeletal radiographic and scintigraphic features. Radiology 171:403–414. Hayirlioglu A, Gokaslan H, Cimsit C, Baysal B. 2009. The importance of severity of artrosis for the reliability of bone mineral density measurement in women. Rheumatol Int 29:371–375. Holm IA, Huang X, Kunkel LM. 1997. Mutational analysis of the PHEX gene in patients with X-linked hypophosphatemic rickets. Am J Hum Genet 60:790–797. Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, Cowell CT, Carpenter TO. 2001. Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 86:3889–3899. Ichikawa S, Traxler EA, Estwick SA, Curry LR, Johnson ML, Sorenson AH, Imel EA, Econs MJ. 2008. Mutational survey of the PHEX gene in patients with X-linked hypophosphatemic rickets. Bone 43:663– 666. Igaki JM, Yamada M, Yamazaki Y, Koto S, Izawa M, Ariyasu D, Suzuki E, Hasegawa H, Hasegawa Y. 2011. High iFGF23 level despite hypophosphatemia is one of the clinical indicators to make diagnosis of XLH. Endocri J 58:647–655. Levy-Litan V, Hershkovitz E, Avizov L, Leventhal N, Bercovich D, ChalifaCaspi V, Manor E, Buriakovsky D, Hadad Y, Goding J, Parvari R. 2010. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene. Am J Hum Genet 86:273–278. Lorenz-Depiereux B, Bastepe M, Benet-Page`s A, Amyere M, Wagenstaller J, Mu¨ller-Barth U, Badenhoop K, Kaiser SM, Rittmaster RS, Shlossberg AH, Olivares JL, Loris C, Ramos FJ, Glorieux F, Vikkula M, Ju¨ppner H, Strom TM. 2006. DMP1 mutations in autosomal recessive hypophosphatemia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 38:1248–1250. Lorenz-Depiereux B, Schnabel D, Tiosano D, Ha¨usler G, Strom TM. 2010. Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal-recessive hypophosphatemic rickets. Am J Hum Genet 86:267–272. Morey M, Castro-Feijo´o L, Barreiro J, Cabanas P, Pombo M, Gil M, Bernabeu I, Dı´az-Grande JM, Rey-Cordo L, Ariceta G, Rica I, Nieto J, Vilalta R, Martorell L, Vila-Cots J, Aleixandre F, Fontalba A, SorianoGuille´n L, Garcı´a-Sagredo JM, Garcı´a-Min˜aur S, Rodrı´guez B, Juaristi S, Garcı´a-Pardos C, Martı´nez-Peinado A, Milla´n JM, Medeira A, Moldovan O, Fernandez A, Loidi L. 2011. Genetic diagnosis of X-linked dominant hypophosphatemic rickets in a cohort study: Tubular reabsorption of phosphate and 1,25(OH)2D serum levels are associated with PHEX mutation type. BMC Med Genet 12:116. Ma¨kitie O, Doria A, Kooh SW, Cole WG, Daneman A, Sochett E. 2003. Early treatment improves growth and biochemical and radiographic outcome in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 88:3591–3597. Quinlan C, Guegan K, Offiah A, O’Neill R, Hiorns MP, Ellard S, Bockenhauer D, Van’t Hoff W, Waters AM. 2012. Growth in PHEX-associated X-linked hypophosphatemic rickets: The importance of early treatment. Pediatr Nephrol 27:581–588. Reid IR, Hardy DC, Murphy WA, Teitelbaum SL, Bergfeld MA, Whyte MP. 1989. X-linked hypophosphatemia: A clinical, biochemical, and histopathological assessment of morbidity in adults. Medicine (Baltimore) 68:336–352. Rosenthall L. 1993. DEXA bone densitometry measurements in adults with X-linked hypophosphatemia. Clin Nucl Med 18:564–566. Ruppe MD, Brosnan PG, Au KS, Tran PX, Dominguez BW, Northrup H. 2011. Mutational analysis of PHEX, FGF23 and DMP1 in a cohort
PEKKARINEN ET AL. of patients with hypophosphatemic rickets. Clin Endocrinol (Oxf) 74: 312–318. Ruppe MD. X-linked hypophosphatemia. In: Pagon RA, Bird TD, Dolan CR, Stephens K, Adam MP, editors. GeneReviewsTM [Internet]. Seattle (WA): University of Washington, Seattle. 2012. [1993-2012 Feb 09 updated 2012 Sep 27]. Saito T, Nishii Y, Yasuda T, Ito N, Suzuki H, Igarashi T, Fukumoto S, Fujita T. 2009. Familial hypophosphatemic rickets caused by a large deletion in PHEX gene. Eur J Endocrinol 161:647–651.
2937 Song HR, Park JW, Cho DY, Yang JH, Yoon HR, Jung SC. 2007. PHEX gene mutations and genotype-phenotype analysis of Korean patients with hypophosphatemic rickets. J Korean Med Sci 22:981– 986. Stickler GB, Beabout JW, Riggs BL. 1970. Vitamin D-resistant rickets: Clinical experience with 41 typical familial hypophosphatemic patients and 2 atypical nonfamilial cases. Mayo Clin Proc 45:197–218. Strom TM, Juppner H. 2008. PHEX, FGF23, DMP1 and beyond. Curr Opin Nephrol Hypertens 17:357–362.
Unusually Severe Hypophosphatemic Rickets Caused by a Novel and Complex Re-Arrangement of the PHEX Gene Tuula Pekkarinen,1* Bettina Lorenz-Depiereux,2 Martina Lohman,3 and Outi Ma¨kitie4,5,6 1
Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, Vantaa, Finland
2
Institute of Human Genetics, Helmholtz Center Munich, Neuherberg, Germany HUS Medical Imaging Center, Helsinki University Hospital, Helsinki, Finland
3 4
Pediatric Endocrinology and Metabolic Bone Diseases, Children’s Hospital, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland 5 Folkha¨lsan Institute of Genetics, Helsinki, Finland 6
Department of Molecular Medicine and Surgery, Karolinska Institutet, and Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden Manuscript Received: 24 March 2014; Manuscript Accepted: 9 July 2014
X-linked hypophosphatemia (XLH) is caused by mutations in PHEX. Several other genetic forms of hypophosphatemia have also been described. These disorders share variable clinical presentation ranging from mild hypophosphatemia to severe lower extremity bowing. We report on a 43-year-old woman with short stature, painful leg deformities, and poor dentation. Her biochemical profile showed hypophosphatemia with renal phosphate wasting. Due to unusually severe clinical presentation and absence of mutations in Sanger sequencing of the PHEX gene, quantitative multiplex ligation-dependent probe amplification was performed. A large deletion within the PHEX gene encompassing exons 8 to 11 was identified. We generated a specific junction fragment using long-range PCR and sequenced the junction fragment to determine the exact deletion breakpoints. We found a heterozygous novel complex re-arrangement involving gross deletions, insertions, and inversion of PHEX (hg19: g.22,115,003_22,141,395del;g:22,145,536_22,150,789delinsCins22, 114,640_22,114,698invinsA). Thus, the complex re-arrangement including a deletion of coding exons 8 to 11 of the PHEX can be regarded as the cause of XLH in the patient reported here. Phosphate and active vitamin D treatment was initiated with subsequent relief in bone pain and physical improvement. This report expands the spectrum of clinical severity underlying genetic defects in XLH and highlights the importance of conventional medical therapy even at adult age. Furthermore, our findings underscore the importance of search for gene deletions in patients with suspected XLH. Ó 2014 Wiley Periodicals, Inc.
Key words: X-linked hypophosphatemia; novel deletions/ insertion in PHEX gene; severe phenotype
INTRODUCTION Hypophosphatemia is an established cause of rickets. The most common inherited form of hypophosphatemic rickets is X-linked
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Pekkarinen T, Lorenz-Depiereux B, Lohman M, Ma¨kitie O. 2014. Unusually severe hypophosphatemic rickets caused by a novel and complex re-arrangement of the PHEX gene. Am J Med Genet Part A 164A:2931–2937.
hypophosphatemia (XLH; OMIM 307800), which results from mutations in the PHEX (phosphate regulating endopeptidase homolog, X-linked) and is inherited in an X-linked dominant pattern [Anonymous, 1995; Carpenter et al., 2011]. A few other genetic forms of hypophosphatemia have also been described. They differ from XLH by their inheritance pattern but clinical course, biochemistry and radiographic features overlap. XLH may result from reduced cleavage and consequently increased concentration of the phosphaturic protein fibroblast growth factor 23 (FGF23), which is made by osteocytes and activates renal KLOTHO/FGFR1 receptor heterodimers to inhibit renal phosphate reabsorption and Conflict of interest: none Grant sponsor: Folkha¨lsan Research Foundation; Grant sponsor: Sigrid Juselius Foundation; Grant sponsor: Academy of Finland; Grant sponsor: Swedish Research Council. Correspondence to: Tuula Pekkarinen, Division of Endocrinology, Department of Medicine, Helsinki University Central Hospital, Sairaalakatu 1, Vantaa, P.O.Box 900, FI-00029 HUS. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 13 August 2014 DOI 10.1002/ajmg.a.36721
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1,25-dihydroxyvitamin D synthesis [Strom and Juppner, 2008]. Gain-of-function mutations in the FGF23 may render it resistant to cleavage and underlie the autosomal dominant form of hypophosphatemic rickets (ADHR, MIM 193100) [ADHR Consortium, 2000]. In addition, two autosomal recessive forms of hypophosphatemic rickets (ARHR1 OMIM 241520, ARHR2 OMIM 613312) have recently been described. These are caused by inactivating mutations in the DMP1 (dentin matrix protein) and ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) respectively, encoding proteins also involved in the regulation of FGF23 [Feng et al., 2006; Lorenz-Depiereux et al., 2006; LorenzDepiereux et al., 2010; Levy-Litan et al., 2010]. The diagnosis of XLH can be challenging. It should be considered if bowing of legs and delayed growth are present in early childhood [Stickler et al., 1970; Holm et al., 2001], but it may be misdiagnosed as nutritional rickets, metaphyseal dysplasia or physiological bowing [Carpenter et al., 2011]. However, early diagnosis is important as treatment during growth partially corrects leg deformities and improves adult height [Ma¨kitie et al., 2003; Quinlan et al., 2012]. Autosomal dominant and recessive hypophosphatemic rickets share common clinical features with XLH but are far rarer and show different inheritance pattern; therefore genetic analysis has been proposed to be mandatory for proper diagnosis of hypophosphatemias [Saito et al., 2009]. During the last two decades, increased knowledge of the various forms of hypophosphatemia has improved diagnostic accuracy and enabled development of treatment guidelines [Carpenter et al., 2011]. We report here on a woman with unusually severe clinically diagnosed hypophosphatemic rickets, which was genetically confirmed as XLH at the age of 43.
CLINICAL REPORT This study was approved by the Research Ethics Board of the Helsinki University Central Hospital, and the patient gave her written informed consent to participation. Because of severe bone pain and leg deformities, this 43-year-old woman of Middle East origin was referred to the Division of Endocrinology, Helsinki University Central Hospital, after her immigration to Finland in 2010. No previous medical records were available, but she described progressive lower limb bowing after age of two. In early childhood, she was treated with intramuscular vitamin D injections in her native country. After age 6, she underwent numerous orthopedic procedures, like osteotomies, followed by immobilization in plaster. Her physical impairment progressed and growth decelerated after these unsuccessful operations. Her family members were of normal height and healthy, and there was no history of parental consanguinity. She complained of bone pain mainly in the legs. She had difficulties to walk and used crutches. She required substantial personal help and assistive equipment to manage everyday life. On physical examination, her height was 127 cm and body weight 47.7 kg. She had four permanent teeth left and used dental prosthesis. Hearing was normal. Right leg was severely deformed, possibly due to previous operations, and amputation was considered. She had coxa vara but knee and hip movements were normal; elbow extension was limited. Laboratory evaluation showed moderate hypophosphatemia and renal phosphate wasting with normal parathyroid hormone level, no hypercalciuria, vitamin D deficiency, and normal renal function (Table I). There was no sign of anemia, coeliac disease or
TABLE I.. Biochemical and Bone Density Findings of the Patient Parameter Ca-ion (mmol/l/pH7.4) Phosphorus (mmol/l) Magnesium (mmol/l) 25(OH) vitamin D (nmol/l) 1,25(OH)2vitamin D (pmol/l) Alkaline phosphatase (U/l) Parathyroid hormone (ng/L) FGF-23 (ng/L) P-Creatinine (mmol/l) P-Albumin (g/l) 24 h U-Ca (mmol/24 h) 24 h U-Pi (mmol/24 h) 24 h U volume (l/24 h) Tubular reabsorption of phosphorusa (mmol/L) U-INTP (nmol/mmol Creatinine) PINP (mg/L) Lumbar spine BMD Z-scorec Femoral neck BMD Z-score right/leftc a
1 year on therapy 1.19 0.54 – 40 100 113 69b – 38 0.65 54.0 2.1 – 40 46.8 – –
Based on 2-hour samples, calculated by use of the equation: serum Pi-(Urine Pi x serum creatinine/urine creatinine). Reference range 12–47 ng/l. Measured by dual-energy X-ray absorptiometry using A Lunar Prodigy scanner (Lunar Radiation Corp., Madison, WI, USA.).
b c
Before therapy 1.15 0.49 0.74 41 124 82 67 62 37 36.0 0.91 17.7 1.1 0.45 67 32.4 þ 2.7 1.9/1.0
2 years on therapy 1.20 0.69 – 52 – – 44b – 35 – 1.01 60.2 1.6 – – – – –
Reference range 1.16 – 1.3 0.76 – 1.41 0.71 – 0.94 > 40 45 – 165 35 – 105 8 – 73 10 – 50 50 – 90 36 – 45 1.3 – 6.5 20 – 50 – – < 65 19 – 84 – –
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Fanconi syndrome, no glucosuria or proteinuria. At the time of referral, she was using daily calcium supplementation (500 mg) with cholecalciferol (10 mg) and etoricoxib for pain. Radiographs showed non-homogeneous bone structure and coarse trabeculation of the metaphyseal regions. The long bones of the lower limbs were short, abnormally shaped and severely bowed. Pseudofractures and enthesopathy were seen (Fig. 1). Periosteal calcification was seen in forearms (Fig. 2). Several teeth were lacking but no hyperostosis was seen in the skull (Fig. 3). The lower lumbar spine showed narrowing of the central spinal canal (Fig. 4). Oral phosphate supplement was commenced with 2250 mg (50 mg/kg) daily divided into four doses; this was combined with vitamin D analogue alfacalcidiol 0.5 mg twice daily and
FIG. 1. Standing weight bearing full-length radiographs of the lower extremities showing severe bowing of bilateral femurs and right tibia. Several insufficiency fractures (Looser’s zones) are seen (arrows). Entesopathy is seen at the insertion areas of Musculus rectus femoris bilaterally ( ). Signs of arthritis of the hips with sclerosis and limited intra-articular spacing are seen.
FIG. 2. A-P-radiograph of the left lower arm shows ossification along the cortexes of the radius diaphysis (arrow).
cholecalciferol 20 mg, later 40 mg daily. Her bone pain diminished within weeks after treatment onset and gait improved; she was able to walk with one elbow crutch and short distances even without assistance. Table I shows laboratory data during treatment. Later,
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FIG. 3. Lateral radiograph of the skull shows lacking upper teeth and only a few lower teeth, but no hyperostosis of the skull.
orthopedic operations to correct lower limb deformities were started. Due to the observed clinical symptoms, we decided to analyze PHEX by capillary sequencing. DNA was extracted from peripheral blood. All the 22 exons and the region around the presumable polyadenylation signal of the PHEX were amplified with intronic primers using PCR and sequenced on both the sense and antisense strand. No mutations within the entire coding region and the splice junctions of the PHEX were identified by Sanger sequencing. As 20– 30% of PHEX mutations are gross deletions [Morey et al., 2011], which span one or more exons and which cannot be detected by PCR and sequencing in female patients, we decided to use a quantitative approach (multiplex ligation-dependent probe amplification MLPA, MRC Holland) to search for dosage differences within the 22 exons. We found a heterozygous deletion of PHEX exons 8–11. We investigated this further by performing long-range PCR with primers in intron 7 and 12 of the PHEX gene to determine the deletion breakpoints. We generated a long-range PCR product of approximately 7 kb, and subsequently performed direct sequencing to identify the deletion breakpoints. Upon identification of the breakpoints, we subjected our findings to BLAT (UCSC Browser), and finally found a complex re-arrangement consisting of a large deletion of 27.7 kb (hg19:g.[22,115,003_22,141,395del(;) ¼ ]; NM_000444.5:c.[850-433_1302 þ 18085del(;) ¼ ], NP_000435.3: p.[Ile284_Met434del(;) ¼ ]) including exons 8–11 of the PHEX coupled by a second smaller proximal deletion/insertion of 5.2 kb. We found a duplication of 58 bp of intron 7 (hg19: g.22,114,640_22,114,698) inverted and inserted within the smaller deletion including two single basepair appendices at the 50 (C) and 30 (A) end of the inversion (hg19:g:22,145,536_22,150,789delins-
FIG. 4. Lateral radiograph of the lumbar spine shows narrowing of the spinal canal in the lower lumbar spine (arrows) due to short pedicles, enlarged facet joints, and spondylosis.
Cins22,114,640_22,114,698invinsA) (Fig. 5). In addition, we were not able to amplify the normal full-length allele of 38.6 kb by long-range PCR.
DISCUSSION We describe a middle-aged hypophosphatemic woman in whom a novel complex re-arrangement of the PHEX was identified. Her phenotype includes poor dentation, short stature, childhood-onset severe lower limb varus deformity requiring osteotomies and causing severe physical impairment with pain in adulthood. Her early treatment, vitamin D injections, was the treatment of choice in XLH 40 years ago, suggesting that she was diagnosed in childhood but did not have access to conventional phosphate and active vitamin D therapy prior to immigration. Reports concerning morbidity of untreated adult patients with a clinical diagnosis of XLH have revealed that symptomatic
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FIG. 5. Location of the complex re-arrangement involving gross deletions, insertions, and inversion of the PHE X gene in the patient.
osteomalacia, enthesopathy, joint pain, dental problems and degenerative joint disease arising from lower limb deformities are common [Reid et al., 1989]. Typical radiographic features in the appendicular skeleton of vitamin D treated adults include abnormally bowed long bones, looser zones due to insufficiency fractures, osteoarthritis in ankles, knees, feet, sacroiliac joints and wrists, abnormally angular (trapezoidal) distal femoral condyles, shortening of talar neck and flattening of the talar dome and accessory ossicles. Other common findings are spinal stenosis and enthesopathy [Hardy et al., 1989]. Our patient demonstrated several of these radiographic findings. A wide variation in the severity and clinical manifestations of untreated XLH patients, even within the same family is typical [Stickler et al., 1970]. Recently, Beck-Nielsen et al. [2010] identified through family screening genetically verified, untreated XLH adults who displayed very few symptoms. Compared with these reports, the presentation of our patient seemed to be unusually severe. Since the 1980’s, childhood-onset treatment with biologically active vitamin D and phosphate has greatly improved bone mineralization and growth of patients with XLH. In adults, medical therapy is empirical and the goal is clinical improvement. Candidates for treatment are adult patients with spontaneous insufficiency fractures, biochemical evidence of osteomalacia and disabling skeletal pain, and those pending orthopedic procedures [Carpenter et al., 2011]. Therapy should start six months prior to the intervention and continue until bone healing is complete. In our patient, treatment initiation at the age of 43 resulted in rapid pain relief and gait improvement, and subsequent orthopedic surgeries were successful. Low serum concentrations of phosphate and 1,25(OH)2 vitamin D and decreased renal phosphate reabsorption observed here are characteristic for XLH. Normal serum alkaline phosphatase was surprising, but it has been reported among adult XLH patients regardless of treatment [Ruppe, 2012]. High serum FGF23 despite hypophosphatemia is one indicator of XLH. Here the FGF23 level was only mildly elevated. Igaki et al. [2011] did not find any correlation between circulating FGF23 levels and measures of
disease severity in XLH, suggesting that the link between XLH severity and serum FGF23 is probably not straightforward. Despite the fact that XLH is a mineralization defect with osteomalacia, elevated z-score of lumbar spine areal bone mineral density was observed in our patient and has been reported previously [Reid et al., 1989; Rosenthall, 1993; Beck-Nielsen et al., 2010; Cheung et al., 2013]. Among adults, extra-osseous calcifications or secondary degenerative findings in the lumbar spine may explain this [Hayirlioglu et al., 2009; Carpenter et al., 2011]. PHEX is a relatively large gene with 22 coding exons. Several mutations have been described, with no mutational hotspots. Studies evaluating genotype–phenotype correlations in XLH have suggested a correlation between more severe bone disease, as determined by the severity of deformity and a history of osteotomies, and truncating mutations [Holm et al., 2001] or mutations in the C-terminal region [Song et al., 2007]. Another study showed that clearly deleterious PHEX mutations (nonsense mutations, insertions or deletions, and splice site mutations leading to premature stop codons) associated with lower tubular phosphate reabsorption and lower calcitriol levels when compared to plausibly deleterious mutations (missense changes or in-frame deletions) [Morey et al., 2011]. Our patient had a previously undescribed complex re-arrangement including a large deletion involving exons 8–11 of the PHEX, and therefore this change is likely to significantly alter the function of the PHEX protein. This report highlights the importance of careful genetic analysis of PHEX with not only normal sequence analysis but with multiplex ligation-dependent probe amplification (MLPA) or comparable methods to detect deletions and/or duplications. Various studies have shown that direct sequencing of the coding regions and intronic borders of PHEX detect mutations in 57–78% of individuals with the clinical and biochemical diagnosis of XLH [Holm et al., 1997; Dixon et al., 1998; Ichikawa et al., 2008; Gaucher et al., 2009; Ruppe et al., 2011]. When both exon sequencing and MLPA analysis are used, mutations can be detected in a significantly larger proportion of patients [Clausmeyer et al., 2009; Morey et al., 2011; Beck-Nielsen et al., 2012].
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CONCLUSION Management of severe bone deformities of XLH in adulthood is highly complex and challenging. Therefore, with this case we highlight the significant clinical variability in XLH, which is further impacted by the timing of medical therapy and success of surgical interventions. Nowadays, early diagnosis enables early initiation of proper treatment which in turn improves height outcome and reduces the need for surgical interventions. The care of XHL patients should be centralized to specialist referral centers. A genetic diagnosis enables appropriate genetic counseling and prenatal or preimplantation diagnostics and should nowadays be regarded a part of diagnostic work-up. In XLH, deletions in the PHEX are frequently observed, and these should be sought when Sanger sequencing fails to detect a causative mutation.
ACKNOWLEDGMENTS We thank Tim Strom for his contribution to this work and Sandy Lo¨secke for excellent assistance. Financial support was provided by grants from Folkha¨lsan Research Foundation, Sigrid Juselius Foundation, the Academy of Finland and the Swedish Research Council.
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