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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 10 3459-3462
Copyright © 1998 by The Endocrine Society

Original Studies

A PHEX Gene Mutation Is Responsible for Adult-Onset Vitamin D-Resistant Hypophosphatemic Osteomalacia: Evidence That the Disorder Is Not a Distinct Entity from X-Linked Hypophosphatemic Rickets1

Michael J. Econs, Nancy E. Friedman, Peter S. N. Rowe, Marcy C. Speer, Fiona Francis, Tim M. Strom, Claudine Oudet, John A. Smith, James T. Ninomiya, Benjamin E. Lee and Heather Bergen

Departments of Medicine (M.J.E.) and Radiology (J.A.S.), Indiana University, Indianapolis, Indiana 46202; Departments of Pediatrics (N.E.F.) and Medicine (M.C.S., B.E.L., H.B.), Duke University, Durham, North Carolina 27710; University College London (P.S.N.R.), London, United Kingdom NW3 2PF; Max-Planck Institut für Molekulare Genetik (F.F.), Berlin, Germany; Abteilung Medizinische Genetik, Kinderpoliklinik der Ludwig Maximilians Universität (T.M.S.), Munich 80336, Germany; IGBMC, Parc d’Innovation (C.O.), 67404 Illkirch, France; and the Department of Orthopedic Surgery, Medical College of Wisconsin (J.T.N.), Wisconsin 53226

Address all correspondence and requests for reprints to: Michael J. Econs, M.D., F.A.C.P., F.A.C.E., Indiana University School of Medicine, 975 W. Walnut Street, IB 445, Indianapolis, Indiana 46202. E-mail: mecons{at}iupui.edu


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Previous investigators described a kindred with an X-linked dominant form of phosphate wasting in which affected children did not have radiographic evidence of rickets, whereas older individuals were progressively disabled by severe bowing. They proposed that this kindred suffered from a distinct disorder that they referred to as adult-onset vitamin D-resistant hypophosphatemic osteomalacia (AVDRR). We recently identified a gene, PHEX, that is responsible for the disorder X-linked hypophosphatemic rickets. To determine whether AVDRR is a distinct form of phosphate wasting, we searched for PHEX mutations in affected members of the original AVDRR kindred. We found that affected individuals have a missense mutation in PHEX exon 16 that results in an amino acid change from leucine to proline in residue 555. Clinical evaluation of individuals from this family indicates that some of these individuals display classic features of X-linked hypophosphatemic rickets, and we were unable to verify progressive bowing in adults. In light of the variability in the clinical spectrum of X-linked hypophosphatemic rickets and the presence of a PHEX mutation in affected members of this kindred, we conclude that there is only one form of X-linked dominant phosphate wasting.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
X-LINKED hypophosphatemic rickets (XLH) is the most common inherited disorder of renal phosphate wasting. It is a dominant disorder that results from mutations in the PHEX1 gene that is located on Xp22.1 (1). Although disease severity is highly variable, affected individuals may present with bone pain, joint stiffness, short stature, tooth abscess, lower extremity deformity, and radiographic evidence of rickets in children (2). Studies by Frymoyer and Hodgkin described a large kindred with an X-linked dominant disorder of isolated phosphate wasting that they termed adult-onset vitamin D-resistant hypophosphatemic osteomalacia (AVDRR) (3). These authors asserted that the disorder was distinct from X-linked hypophosphatemic rickets because affected children (n = 13) did not have radiographic evidence of rickets, and patients typically presented with clinical manifestations of the disease in the forth or fifth decade of life (3). Although studies performed by our group established that radiographic evidence of rickets is not an invariant feature of XLH, the majority of affected children have radiographic evidence of rickets (4). Thus, the possibility exists that there are two forms of X-linked dominant hypophosphatemic rickets due to mutations of two different genes on the X-chromosome. In light of recent observations that XLH results from mutations in the PHEX gene, we sought to determine whether a PHEX mutation was present in the original AVDRR kindred described by Frymoyer and Hodgkin. Our results suggest that there is only one form of X-linked hypophosphatemic rickets.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

We clinically evaluated and obtained blood from 85 members of the original Vermont kindred, which is of French-Scottish ancestry (3). The criteria for determining affection status were previously described (5). In brief, affected individuals were defined by the presence of hypophosphatemia, normocalcemia, and normal renal function. Serum phosphorus values for children were interpreted using age-specific normal ranges (6). Tubular maximum reabsorption of phosphate per glomerular filtration rate was calculated according to the nomogram of Bijvoet and Walton (7). The study was approved by the Duke University Medical Center institutional review board, and all subjects and/or their parents gave written informed consent before participating in the studies.

Sequencing of genomic DNA

DNA extraction from whole blood was performed as previously described (8). Primers pairs were developed for each of the 22 PHEX gene exons as previously described (9). Thirty to 60 ng genomic DNA were PCR amplified with exon-specific primers. PHEX exon 16 amplification was performed in a total reaction volume of 30 µL containing 2.0 mmol/L MgCl2; 100 mmol/L KCl; 10 mmol/L Tris-HCl (pH 8.3); 5 mmol/L NH4Cl; 200 µmol/L each of deoxy (d)-ATP, dGTP, dCTP, and dTTP; 120 ng intronic primers (B8F1, 5'-AGG TAC TCA TCA TTG AAT CAA TCT; B8R1, 5'-ATG TTC TTC CTA ATT GGT CAG TAA); and 0.9 U AmpliTaq polymerase (Perkin-Elmer Corp., Branchburg, NJ). PCR temperatures were 94, 60, and 72 C for 1 min each for 36 cycles, followed by 9 min at 72 C. The resulting products were column purified with Sephadex G-50 (coarse; Pharmacia, Uppsala, Sweden). Sequencing was performed with 33P using the ThermoSequenase kit (Amersham, Cleveland, OH) in accord with the manufacturer’s specifications. Mutations were confirmed by reamplification of genomic DNA and sequencing in both forward and reverse directions.

Single stranded conformational polymorphism (SSCP) methods

SSCP (10) was performed after genomic DNA was amplified with the exon 16-specific primers under the conditions noted above, except that the total reaction volume was 15 µl. The final product was diluted 1:1 with SSCP stop dye (95% formamide, 10 mmol/L NaOH, 0.1% xylene cyanol, and 0.1% bromophenol blue), and 4 µL were heated to 95 C and subjected to electrophoresis on a 6.5% polyacrylamide gel with 5% glycerol. Electrophoresis was performed overnight at 6 watts in 0.5 x TBE.

Protein structure analysis

We performed secondary structure predictions with the GCG peptide structure computer program (11).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
We sequenced genomic DNA from affected pedigree members and controls to look for PHEX mutations. Sequences from several affected members of the kindred indicate that they have a T to C transition in PHEX exon 16 that results in an amino acid change from leucine to proline in residue 555 (Fig. 1Go). To exclude the possibility that this change was a simple polymorphism, we analyzed genomic DNA from affected individuals, 99 female and 73 male Caucasian controls (271 normal chromosomes), using SSCP. Aberrant bands were only seen from affected kindred members, and the mutation segregated with the disease within the family (Fig. 2Go).



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  		Figure 1. Sequence of PHEX exon 16 from an affected male (the father of patient 1), patient 1, and a normal control. This sequence was performed with the antisense primer. Nucleotide lanes are labeled with the complementary nucleotide from the sense strand to facilitate reading the sequence. Results were unchanged when sequencing was performed with the sense primer (not shown).

 


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  		Figure 2. Family tree with SSCP underneath. The aberrant band segregates with the disease in the family. Closed symbols indicate an affected individual. Open symbols indicate a normal individual. Squares are males; circles are females.

 
Computer analysis with the GCG peptide structure program indicates that the change in primary sequence results in a small, but significant, change in predicted secondary structure. Although the overall transmembranous score is unaltered, and the 5'-hydrophobic region is intact, the mutant form has a slight decrease in hydrophobicity. More importantly, the leucine to proline change at residue 555 results in the appearance of an additional flexible node in the mutant structure and a consequent increased flexibility (Fig. 3Go). This region is adjacent (N-terminal) to the zinc binding motif, and the increased flexibility is predicted to result in major disruption of the catalytic site. Of note, leucine 555 is conserved in other M13 zinc metallopeptidases including neutral endopeptidase, the Kell antigen, and endothelin converting enzyme-1 (9).



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  		Figure 3. A and B, Flexibility plots illustrating the comparative secondary structure profiles for mutant (A) and control (B) PHEX. The curved line represents the primary amino acid sequence. Regions of predicted glycosylation are presented as circles on stalks, and flexible nodes with an index greater than 1.04 are superimposed over the primary sequence (15 16 17 ). The change in sequence at residue 555 results in the appearance of an additional flexible node (arrow) in the mutant structure and consequent increased flexibility. This region is adjacent (N-terminal) to the zinc binding motif, and the increased flexibility is predicted to result in major disruption of the catalytic site. The Chou-Fasman secondary structure prediction and flexibility indexes (15 16 ) were calculated using the GCG peptide structure software (17 ).

 
As it is possible that the observed mutation results in a milder phenotype than has been seen with other mutations, particularly with regard to radiographic evidence of rickets, we studied several members of the original kindred (3). Radiographs of the knees were available from six affected children under 15 yr of age (aged 1.4, 2.5, 8.9, 11.1, 11.6, and 13 yr). Plain films for one child (patient 1, below) demonstrated radiographic evidence of rickets, three (aged 1.4, 8.9, and 13 yr) were suspicious for rickets, and two (aged 11.1 and 11.6 yr) did not display features of rickets. One 2.5-yr-old girl was evaluated at the Duke General Clinical Research Center, and her case report is presented below:

Patient 1

The patient was the 7-lb, 3-oz. produce of a normal pregnancy, labor, and delivery. She was noted to have in-turning of her right foot at birth. She was thought to have physiological bowing initially; however, at 1.5 yr of age, she was noted to have a low phosphorus for age (3.5 mg/dL) but at that time her bowing was not severe. Over the next few months, her bowing increased. We evaluated her at 2.5 yr of age. At that time her height was 89.3 cm (75th percentile), and her weight was 12.9 kg (50th percentile). The serum calcium level was 9.2 mg/dL. The serum phosphorus level was 2.96 mg/dL. The intact PTH level was 24 ng/dL (normal, 15–50), and the 1,25-dihydroxyvitamin D level was 15 pg/mL (normal, 15–50). The serum 25-hydroxyvitamin D3 level was 37 ng/dL (normal, 15–80). Urinary calcium excretion was 0.11 mg/mg creatinine. The serum bicarbonate level was normal, and urinalysis revealed normal pH and concentrating ability. The tubular maximum reabsorption of phosphate per glomerular filtration rate was 2.9. Radiographs revealed rickets in her knees (Fig. 4Go). She was started on calcitriol and phosphorus therapy and did well on therapy, maintaining normal growth. She developed a genu valgus deformity that remained stable.



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  		Figure 4. Radiographs of the lower extremities (A) with detail view (B) of patient 1 at 2.5 yr of age. Note bilateral bowing and radiographic features of rickets, including splaying and irregular calcification at the growth plate.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the current investigation, we examined a kindred that had been reported by previous investigators (3) to have an X-linked dominant phosphate-wasting disorder that was distinct from XLH. They based this contention largely on their observations that hypophosphatemic children from this kindred did not display radiographic evidence of rickets, and clinical deformity was only noted in affected family members who were in the forth or fifth decade of life. Thus, the possibility existed that there were two genes on the X-chromosome that play important roles in phosphate homeostasis. Our results, which show a mutation in PHEX exon 16, demonstrate that the disease in this kindred is not due to a mutation of a second phosphate-regulating gene on the X-chromosome.

Our results confirm and extend our earlier work, which established that radiographic evidence of rickets is not an invariant feature of XLH and that the radiographic features of rickets seen in XLH patients tend to be milder than those in other forms of rickets (4). Our results differ from those of Frymoyer and Hodgkin, as some, but not all, of the affected children in this kindred had radiographic findings that were either suspicious for rickets or clearly demonstrated rickets. Additionally, we were unable to confirm adult onset of lower extremity deformity (Econs, M. J., unpublished observations), and in contrast to the previous study, we found individuals who presented with lower extremity deformity during childhood. It is possible that some of the difference between our observations and those of Frymoyer and Hodgkin is attributable to differences in the ages of the individuals studied, as rickets may be more easily detected in children between the ages of 1.5–6 yr, who are rapidly growing and weight bearing. Unfortunately, these investigators did not provide the ages of the children studied in their report.

It is possible that the leucine to proline mutation observed in this kindred results in a milder or atypical phenotype compared to other PHEX mutations. However, our evaluation of affected kindred members indicates that at least some of these individuals manifest typical features of XLH. Although this is the first report of a leucine to proline mutation in PHEX exon 16, the mutation observed in this family is not atypical. We identified leucine to proline (9) and proline to leucine (9, 11) mutations in other PHEX exons in several XLH kindreds, as have other investigators (12, 13, 14). This is not surprising in light of the significant changes in secondary structure induced by substituting proline for leucine.

It is of great interest that there is substantial variability in the clinical presentation of patients with XLH even when they are from the same family. The reason for this observed variability in disease severity is probably due to differences in the ability to compensate for the genetic abnormality, and this indicates that there are other modifying genes or environmental influences that affect the severity of the disease. Of note, several investigators (9, 11, 12, 13, 14) have found numerous PHEX mutations in XLH patients, and there is no one predominant mutation. In light of the variability in clinical presentation between individuals in the same family, great caution should be exercised when attempting to make genotype/phenotype correlations between families, particularly when a small number of individuals with a particular mutation is available. In any event, our results demonstrate a PHEX gene mutation in affected members of this kindred, and we conclude that there is only one form of X-linked dominant phosphate wasting.


   Acknowledgments
 
The authors thank Dr. John Frymoyer for providing access to this family, and Kristi Viles for technical assistance.


   Footnotes
 
1 Presented in part at the annual scientific meeting of the American Society for Bone and Mineral Research, September 10–14, 1997. This work was supported by Grants AR-42228, AR-27032, MO1-RR-30, and NS-26630. PHEX was previously referred to as PEX; however, the gene symbol has been changed in accord with recommendations from the HUGO/GDB Nomenclature Committee. Back

Received March 27, 1988.

Revised June 22, 1998.

Accepted July 1, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. HYP Consortium. 1995 Positional cloning of PEX: a gene with homologies to endopeptidases is mutated in patients with X-linked hypophosphatemic rickets. Nat Genet. 11:130–136.[CrossRef][Medline]
  2. Econs MJ, Drezner MK. 1992 Bone disease resulting from inherited disorders of renal tubule transport and vitamin D metabolism. In: Favus MJ, Coe FL, eds. Disorders of bone and mineral metabolism. New York: Raven Press; 935–950.
  3. Frymoyer JW, Hodgkin W. 1977 Adult-onset vitamin D-resistant hypophosphatemic osteomalacia. J Bone Joint Surg. 59:101–106.[Abstract/Free Full Text]
  4. Econs MJ, Feussner JR, Samsa GP, et al. 1991 X-linked hypophosphatemic rickets without "rickets." Skel Radiol. 20:109–114.[Medline]
  5. Econs MJ, Pericak-Vance MA, Betz H, Bartlett RJ, Speer MC, Drezner MK. 1990 The human glycine receptor: a new probe that is linked to the X-linked hypophosphatemic rickets gene. Genomics. 7:439–441.[CrossRef][Medline]
  6. Greenberg BG, Winters RG, Graham JB. 1960 The normal range of serum inorganic phosphorus and its utility as a discriminant in the diagnosis in congenital hypophosphatemia. J Clin Endocrinol Metab. 20:364–379.
  7. Walton RJ, Bijvoet OLM. 1973 Nomogram for derivation of renal threshold phosphate concentration. Lancet. 2:309–310.
  8. Econs MJ, Fain PR, Norman M, et al. 1993 Flanking markers define the X-linked hypophosphatemic rickets gene locus. J Bone Miner Res. 8:1149–1152.[Medline]
  9. Rowe PSN, Oudet C, Francis F, et al. 1997 Distribution of mutations in the PEX gene in families with X-linked hypophosphatemic rickets (HYP). Hum Mol Genet. 6:539–549.[Abstract/Free Full Text]
  10. Orita M, Suzuki Y, Sekiya T, Hayashi K. 1989 Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics. 5:874–879.[CrossRef][Medline]
  11. Francis F, Strom TM, Hennig S, et al. 1997 Genomic organization of the human PEX gene mutated in X-linked dominant hypophosphatemic rickets. Genome Res. 7:573–585.[Abstract/Free Full Text]
  12. Holm IA, Huang X, Kunkel LM. 1997 Mutational analysis of the PEX gene in patients with X-linked hypophosphatemic rickets. Am J Hum Genet. 60:790–797.[Medline]
  13. Dixon PH, Wooding C, Trump D, Schlessinger D, Whyte MP, Thakker RV. 1996 Seven novel mutations in the PEX gene indicate molecular heterogeneity for X-linked hypophosphatemic rickets [Abstract]. J Bone Miner Res. 11:S136.
  14. Dixon PH, Wooding C, Christie P, et al. 1997 Mutations of the PEX regulatory and C-terminal regions cause X-linked hypophosphatemic rickets [Abstract]. J Bone Miner Res. 12:S128.
  15. Chou PY, Fasman GD. 1978 Empirical predictions of protein conformation. Annu Rev Biochem. 47:251–276.[CrossRef][Medline]
  16. Chou PY, Fasman GD. 1978 Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 47:45–148.[Medline]
  17. Rice P. 1995 Programme manual for the EGCG package. Cambridge: Hinxton Hall; Human Genome Mapping Project: http://www.hgmp.mrc.ac.uk/homepage.html.



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