Autosomal recessive hypophosphataemic rickets with hypercalciuria is not caused by mutations in the type II renal sodium/phosphate cotransporter gene
Lambert van den Heuvel,1,
Katja Op de Koul1,
Edwin Knots2,
Nine Knoers3 and
Leo Monnens1
1 Department of Pediatrics, University Hospital Nijmegen, Nijmegen,
2 Department of Pediatrics, St Catharine Hospital, Eindhoven,
3 Department of Human Genetics, University Hospital Nijmegen, The Netherlands
 |
Abstract
|
---|
Background. At present the genetic defect for autosomal recessive and autosomal dominant hypophosphataemic rickets with hypercalciuria (HHRH) is unknown. Type II sodium/phosphate cotransporter (NPT2) gene is a serious candidate for being the causative gene in either or both autosomal recessive and autosomal dominant HHRH. In the present study we tested this hypothesis in one autosomal recessive family.
Methods. The gene structure of human NPT2 is known. We tested the complete open reading frame in the affected siblings by polymerase chain reaction in combination with automatic DNA sequencing for the presence of mutations.
Results. We did not observe disease-causing mutations in the NPT2 gene of the affected siblings. A T855C polymorphism resulting in a histidine to arginine transition was present in the open reading frame of NPT2. The polymorphism was present in both affected as well as unaffected family members.
Conclusion. The hypothesis that a defect in the NPT2 gene could be an underlying cause for autosomal recessive HHRH could not be sustained in our study.
Keywords: hypophosphataemic rickets with hypercalciuria; mutation detection; sodium/phosphate cotransporter gene type II
 |
Introduction
|
---|
In recent years it has become increasingly clear that several genetically and phenotypically distinct forms of hereditary hypophosphataemia associated with increased urinary phosphate excretion can be distinguished. The most prevalent disorder is X-linked dominant hypophosphataemia, which is caused by mutations in the PHEX gene [1]. Another variant, less frequently occurring, is hereditary hypophosphataemic rickets with hypercalciuria (HHRH). This disorder is associated with increased levels of 1,25 dihydroxyvitamin D and suppressed parathyroid hormone levels. Both autosomal recessive and autosomal dominant types of HHRH have been described [2]. As yet, the genetic abnormalities underlying HHRH are unknown.
Reabsorption of phosphate in the renal proximal tubule is of critical importance for phosphate homeostasis. It has been shown by physiological studies that the type II sodium/phosphate cotransporter (NPT2) in the apical brush border membrane of proximal tubule cells is the key player in renal reabsorption of phosphate (reviewed in [3]). In mice with targeted inactivation of NPT2, biochemical abnormalities reminiscent of HHRH have been observed [4]. This finding rendered the NPT2 gene a serious candidate for being the causative gene in either or both autosomal recessive and autosomal dominant HHRH.
Since the gene structure of the human NPT2 gene is known [5], we were able to test this hypothesis by screening the coding region of the NPT2 gene for mutations in patients from an autosomal recessive HHRH kindred. Since we did not find any mutations in our patients, we speculate that it is rather unlikely that the NPT2 gene is the causative gene for autosomal recessive HHRH.
 |
Subjects and methods
|
---|
Patients
The pedigree of the family is shown in Figure 1
. Based on consanguinity (I : 1 and I : 2 are first cousins), normal phosphate homeostasis in both parents and presence of HHRH in two of the five sibs, the most likely mode of inheritance is autosomal recessive. The relevant laboratory data of the family members are presented in Table 1
. Both affected children showed hypophosphataemia due to a lowered tubular reabsorption, increased levels of 1,25 (OH)2 D, and lowered parathyroid hormone concentration accompanied with hypercalciuria. Metaphyseal abnormalities of rickets were observed in both.

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 1. Pedigree of family with autosomal recessive HHRH. The parents (I : 1 and I : 2) are first cousins. Symbols: and are affected patients.
|
|
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. We designed primers to ampify all 13 exons and flanking intronic splice sites of the human NPT2 from genomic DNA by PCR (Table 2
). Polymerase chain reaction (PCR) conditions were optimized for each primer pair and 35 cycles of PCR amplification were performed in a standard PCR buffer containing 50 ng of each primer, 0.25 mmol/l dNTPs, 1.5 mmol/l MgCl2, 0.5 U Taq polymerase and PCR buffer (Life Technologies, The Netherlands). PCR products were purified from agarose gel (Qiagen, The Netherlands) and sequenced for both strands by using dyedeoxy terminators on an automated fluorescence-based Applied Biosystems ABI 377 DNA sequencing system. The cDNA sequence and the amino acids were numbered according to Gen Bank accession number L13258.
 |
Results
|
---|
The availability of the human NPT2 gene structure enabled us to screen this gene for mutations in both affected siblings. DNA sequencing of the total coding region of the NPT2 gene did not result in the detection of disease-causing mutations. We observed a T855C polymorphism in the NPT2 open reading frame. This transition changes codon 258 from a histidine to an arginine. We were able to confirm the substitution by means of restriction enzyme analysis with Nco I, which spliced the wildtype amplimers for exon 7 once into two fragments of 174 and 117 bp and does not cut the mutant allele (291 bp). Both index patients as well as two unaffected siblings and the parents were heterozygous for the polymorphism (Figure 2
). Restriction enzyme analysis with Nco I and the exon 7 amplimers of the NPT2 gene revealed an allelic frequency of 86% thymine and 14% cytosine in 50 control alleles.

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 2. Agarose gel (3.5%) electrophoresis of the amplimers of exon 7 after digestion with NcoI. Lane 1, father (I : 1); lane 2, mother (I : 2); lane 3, II : 1 (healthy sib); lane 4, II : 2 (affected sib); lane 5, II : 3 (healthy sib); lane 6, II : 4 (affected sib).
|
|
 |
Discussion
|
---|
The hypothesis that a defect in the type II sodium/phosphate cotransporter could be an underlying cause for HHRH could not be sustained in our study. The absence of mutations in the coding region of the NPT2 gene in our patients makes it very unlikely that NPT2 is the gene causing autosomal recessive HHRH. We did not directly rule out the presence of mutations in the non-coding regions of the NPT2 gene. Nevertheless, the identification of the T855C polymorphism in this gene in a heterozygous and not homozygous state in both patients from this consanguineous family indirectly argues against any causative role of NPT2 in recessive HHRH.
The exclusion of NPT2 as the disease gene, however, does not rule out the possibility that NPT2 is functionally disturbed in our patients [3]. It is possible that, secondary to the as yet unknown primary defect, the expression of NPT2 is downregulated, resulting in decreased proximal tubular reabsorption of phosphate and hypophosphataemia. Such diminished NPT2 expression has been demonstrated, for example, in Hyp and Gy mice, in which hypophosphataemia is caused by mutations in the X-linked PHEX gene.
Based on the proteolytic properties of PHEX, it has been suggested that PHEX is involved in the processing of a humorally-mediated factor that controls the expression of NPT2. Consequently, mutations in PHEX could result in inappropriate degradation of this humoral factor and subsequent decrease of NPT2 expression. It is imaginable that a similar mechanism is responsible for the lowered tubular phosphate reabsorption in our patients with autosomal recessive HHRH.
Alternatively, but less likely, the defect in our patients could be the result of mutations in the type I sodiumphosphate cotransporter (NPT1) gene. This transporter is expressed in the straight part of the proximal tubule but not in the convoluted proximal tubule, the main site of the phosphate reabsorption [7].
In conclusion, we were unable to prove that the most likely candidate for the cause of HHRH, the type II Na/Pi cotransporter gene, is indeed primarily involved in this disorder, at least in the recessive form of HHRH. Whether it causes the dominant type remains to be elucidated. The identification of the primary genetic defect(s) in HHRH will help to further unravel the complex physiology of phosphate transport in the proximal renal tubule.
 |
Notes
|
---|
Correspondence and offprint requests to: Dr L. P. van den Heuvel, Department of Pediatrics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. 
 |
References
|
---|
-
Econs MJ, Francis F. Positional cloning of the PEX gene: new insights into the pathophysiology of X-linked hypophosphatemic rickets. Am J Physiol1997; 273: F480498
-
Tieder M, Modal D, Shaked U et al. Idiopathic hypercalciuria and hereditary hypophosphatemic rickets. Two phenotypical expressions of a common genetic defect. N Engl J Med1987; 316: 125129[Abstract]
-
Murer H, Forster J, Hilfiker H et al. Cellular/molecular control of renal Na/Pi cotransport. Kidney Int1998; 53 [Suppl 65]: S210
-
Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H, Tenhouse HS. Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal anomalies. Proc Natl Acad Sci USA1998; 95: 53725377[Abstract/Free Full Text]
-
Taketani Y, Miyamoto K, Tanaka K et al. Gene structure and functional analysis of the human Na+ phosphate cotransporter. Biochem J1997; 324: 927934[ISI][Medline]
-
Kruse K, Kracht U, Gopfert G. Renal threshold phosphate concentrations. Arch Dis Child1982; 57: 217223[Abstract]
-
Takeda E, Taketani Y, Morita K, Miyamoto KI. Sodium-dependent phosphate cotransporters. Int J Biochem Cell Biol1999; 31: 377381[ISI][Medline]
Received for publication: 30. 7.99
Revision received 30. 7.00.