INVITED REVIEW
Positional cloning of the PEX gene: new insights into the pathophysiology of X-linked hypophosphatemic rickets

Michael J. Econs1 and Fiona Francis2

1 Department of Medicine, Duke University Medical Center and the Durham Veterans Affairs Medical Center, Durham, North Carolina 27710; and 2 Max-Planck Institut für Molekulare Genetik, 14195 Berlin, Germany

    ABSTRACT
Top
Abstract
Introduction
Conclusion
References

X-linked hypophosphatemic rickets (HYP) is the most common form of hereditary renal phosphate wasting. The hallmarks of this disease are isolated renal phosphate wasting with inappropriately normal calcitriol concentrations and a mineralization defect in bone. Studies in the Hyp mouse, one of the murine models of the human disease, suggest that there is an ~50% decrease in both message and protein of NPT-2, the predominant sodium-phosphate cotransporter in the proximal tubule. However, human NPT-2 maps to chromosome 5q35, indicating that it is not the disease gene. Positional cloning studies have led to the identification of a gene, PEX, which is responsible for the disorder. Further studies have led to identification of the murine Pex gene, which is mutated in the murine models of the disorder. These studies, in concert with other studies, have led to improved understanding of the pathophysiology of HYP and a new appreciation for the complexity of normal phosphate homeostasis.

hypophosphatemia; osteomalacia; endopeptidase; X chromosome

    INTRODUCTION
Top
Abstract
Introduction
Conclusion
References

THERE ARE SEVERAL hereditary disorders that result in isolated renal phosphate wasting, including X-linked hypophosphatemic rickets (HYP), autosomal dominant hypophosphatemic rickets (ADHR), and hereditary hypophosphatemic rickets with hypercalciuria (HHRH). The genes mutated in these diseases are likely to play important roles in phosphate homeostasis. The existence of multiple forms of hereditary phosphate wasting indicates that control of phosphate homeostasis is a complex process. Understanding the pathogenesis of these disorders will provide insight into this process and may lead to improved therapies for these conditions. This review will focus on recent developments in understanding the pathogenesis of HYP, which is the most common inherited disorder of renal phosphate wasting.

HYP is an X-linked dominant disorder with a prevalence of ~1:20,000. Patients may present with lower extremity deformities, rickets, short stature, bone pain, dental abscesses, enthesopathy, and osteomalacia (22). However, severity of the phenotype varies considerably. Indeed, affected members of the same family may have markedly different phenotypes, and some individuals have only minimal symptoms. Although there is controversy about whether there is a gene dosage effect, recent evidence suggests that the disorder is a classic dominant condition with equal severity in males and females (89). The hallmark of the disorder is renal phosphate wasting with resulting hypophosphatemia. Patients also have inappropriately normal concentrations of calcitriol (16, 52, 75, 77).

    MURINE MODELS

Many insights into the pathogenesis of HYP have been derived from studies of two murine homologs of the human disease, Hyp and Gy mice. Linkage studies in the mouse have mapped the Hyp and Gy mutations to a region of the mouse X chromosome syntenic to the human HYP locus (27, 39, 54, 78). The Hyp mouse arose as a spontaneous mutation and has been bred on the C57BL/6J background (27). The Gy mutation was induced by irradiation, and these mice have been bred on the B6C3H background (54). Both murine models have renal phosphate wasting, impaired mineralization, and growth retardation. However, the Gy mouse also has inner ear abnormalities, deafness, hyperactivity, and circling behavior, and the male Gy mouse does not survive on the C57BL/6J background (58). Although both mice have been studied, more experiments have been performed with Hyp mice. Controversy exists regarding alleged biochemical differences between the two mouse models (see below). Also, in accordance with the human disease, there are not marked differences between male and female Hyp mice (66); however, fewer data are available for Gy mice.

    PATHOGENESIS OF PHOSPHATE WASTING

Since parathyroid hormone (PTH) is known to prevent reabsorption of phosphate in the kidney, one might expect it to be involved in the pathogenesis of HYP. However, several lines of evidence suggest that this is not the case. First, PTH concentrations are normal in HYP patients (2); second, parathyroidectomy does not alleviate the phosphate wasting in Hyp mice (11); and third, Lyles et al. (51) described a HYP patient with concurrent idiopathic hypoparathyroidism who had phosphate wasting once serum calcium levels were corrected. Hence, it is generally believed that PTH is not responsible for hypophosphatemia in HYP, and researchers have pursued other lines of investigation to determine the pathogenesis of phosphate wasting.

In this regard, studies done in the Hyp mouse demonstrate that the renal phosphate wasting results from decreased sodium-dependent phosphate transport in the brush-border membrane of the renal proximal tubule (83). Subsequent studies have shown that, although the low-affinity/high-capacity transport mechanism is intact, there is a defect in the high-affinity/low-capacity transporter (81). Indeed, the maximal transport rate (Vmax) is about one-half of normal with no change in affinity for phosphate, a finding consistent with a decreased transporter number in the Hyp mouse. This high-affinity/low-capacity sodium-dependent phosphate cotransporter (Npt-2) has recently been cloned (88), and Hyp mice have been shown to have an ~50% decrease in Npt-2 mRNA and protein (9, 84). Studies done in the Gy mouse also demonstrate a reduction in Vmax of the high-affinity/low-capacity transport system (82), and a decrease in Npt-2 mRNA and protein was also observed by Tenenhouse et al. (80). Conversely, Collins and Ghishan (10) have reported that Hyp and Gy mice differ in this regard, since they found normal levels of Npt-2 mRNA in Gy mice, with decreased levels of Npt-2 protein. The localization of human NPT-2 on chromosome 5q35 eliminated it as a candidate gene for HYP (40). However, the data suggest that the HYP gene is involved in regulation of NPT-2 expression.

Despite the above results, it remained unclear whether the phosphate wasting resulted from a primary renal defect or whether it resulted from elaboration of a humoral factor that alters phosphate uptake in the renal proximal tubule. To test the possibility of a humoral abnormality, Meyer et al. (57) performed parabiosis between Hyp and normal mice. They found that normal mice joined to Hyp mice had a progressive reduction in plasma phosphate over 3 wk and that these mice had a greater renal phosphate excretion index than normal mice joined to other normal mice. Furthermore, after the normal/Hyp pairs were separated, plasma phosphate returned to normal levels in the normal mouse within 24 h. At 2 and 7 days these normal mice had "rebound hyperphosphatemia" compared with mice separated from normal/normal pairs (57). As exciting as these findings were, parabiosis studies have significant limitations. Urine creatinine concentration in normal mice parabiosed to Hyp mice was almost twice as high as that in normal mice joined to normal mice. Since urine volume and/or plasma creatinine were not measured, it is difficult to determine whether renal function was altered, although it is likely that these mice simply had more concentrated urine than normal to normal parabiotic pairs. Additionally, some of the fall in plasma phosphate may have been secondary to inanition, since plasma phosphate fell in normal mice joined to normal mice but not as much as the fall in phosphate when normal mice were joined to Hyp mice.

To avoid some of the limitations of parabiosis experiments, Nesbitt et al. (61) performed renal cross-transplantation studies in normal and Hyp mice. When normal kidneys were transplanted into nephrectomized Hyp mice, the kidneys wasted phosphorus. When Hyp kidneys were transplanted into nephrectomized normal mice, the kidneys retained phosphorus normally. Thus the defect in the Hyp mouse is neither corrected nor transferred by renal cross-transplantation, and the phosphate transport defect in the Hyp mouse is not due to an intrinsic renal abnormality. These data have been further confirmed by subsequent tissue culture studies that employed SV40-transformed cells from the S1 segment of the renal proximal tubule from normal and Hyp mice. These studies demonstrate that sodium-dependent phosphate transport in S1 proximal tubule cells is not different between cells obtained from Hyp and normal mice (62). Similar studies have been performed with SV40-transformed proximal tubular cells from Gy and normal mice. These studies also show equivalent phosphate transport between mutant and normal cells (63).

The above data are supported by a case report by Morgan et al. (60), in which a patient with probable sporadic HYP developed renal failure. The patient received a living related renal transplant from an apparently normal sister and redeveloped renal phosphate wasting. Unfortunately, these results are clouded by the observation that ~32% of renal transplant patients have some degree of renal phosphate wasting (68).

Although the above studies established that the defect in Hyp mice is not in the kidney, these studies did not determine alternative tissue(s) that contain the defect. Since bone from Hyp mice does not mineralize normally, some investigators have focused their efforts on studying osteoblasts from the Hyp mouse. An intrinsic osteoblast defect was proposed by Ecarot et al. (18-20) who transplanted periostea and osteoblasts from normal and Hyp mice into the gluteal muscles of normal and Hyp mice. As expected, when normal cells were transplanted into Hyp mice, mineralization was impaired. However, when Hyp cells were transplanted into normal mice, reduction but not normalization of the defect was observed. Although these studies supported the hypothesis that there is a primary osteoblast defect in the Hyp mouse, they did not exclude the possibility that the putative circulating factor in the Hyp mouse could have led to an irreversible developmental defect in the osteoblast. Such a situation would be analogous to the permanent developmental defects that are produced in developing neural tissue by lack of sufficient thyroid hormone at a critical stage of development (7).

Further evidence that the Hyp mouse elaborates a humoral factor causing the phosphate wasting and that the osteoblast plays a role in the pathogenesis of the Hyp defect is provided by the studies of Lajeunesse et al. (43). These investigators found that Hyp serum, when added to the culture media for at least 24 h, impaired phosphate transport in primary mouse proximal tubule cultures (MPTC). This inhibition occurred in a dose-dependent fashion. Additionally, conditioned media from Hyp osteoblasts, but not normal osteoblasts, inhibited phosphate transport in the MPTC when the incubation period was at least 24 h. These data suggest that the Hyp osteoblast is responsible for the release and/or modification of a humoral factor(s) that inhibits phosphate reabsorption (43). It should be noted, however, that these data do not exclude other tissues as important contributors to the pathogenesis of the phosphate wasting and other phenotypic features of the disorder.

The existence of a humoral factor that can impair renal proximal tubular phosphate transport is supported by the existence of tumor-induced osteomalacia. These tumors, which are frequently of mesenchymal origin, result in isolated renal phosphate wasting and inappropriately low serum calcitriol concentrations, both of which resolve when the tumor is removed. Unfortunately, this factor(s), which we have referred to as "phosphatonin," has only been partially purified (6, 23). Since tumors frequently secrete, in abnormal amounts and in an unregulated fashion, substances that have a role in normal physiology, it is plausible that the phosphate wasting observed in patients who have tumor-induced osteomalacia is due to overproduction of a factor(s) that normally controls renal phosphate reabsorption. If such a factor exists, then its role in the phosphate wasting seen in HYP has yet to be determined.

    VITAMIN D METABOLISM

In addition to the phosphate-wasting defect seen in HYP, defects in vitamin D metabolism are postulated. Indeed, treatment of HYP patients with phosphate alone does not result in resolution of the osteomalacia (53). A combination of phosphate and high-dose calcitriol is required (31, 33). In the setting of hypophosphatemia, elevated calcitriol levels are anticipated, since hypophosphatemia increases calcitriol concentrations in both animals and humans (34, 37, 65). Several investigators have instead found normal serum calcitriol concentrations in HYP patients (16, 52, 75, 77). Thus HYP patients have a relative insufficiency of calcitriol in light of their hypophosphatemia. Studies in the Hyp mouse confirm and expand the observations made in humans (49, 56). Lobaugh and Drezner (49) studied the activity of renal 25-hydroxyvitamin D-1alpha -hydroxylase [25(OH)D-1alpha -hydroxylase] in Hyp, normal, and phosphate-depleted mice. This enzyme converts 25(OH)D to 1alpha ,25-dihydroxyvitamin D [1,25(OH)2D], the active form. Although normal mice on phosphate-depleted diets had profoundly increased 25(OH)D-1alpha -hydroxylase activity compared with normal mice on the control diet, 25(OH)D-1alpha -hydroxylase activity in Hyp mice was lower than phosphate-depleted controls, despite similar serum phosphate concentrations. Moreover, 24-hydroxylase is increased in Hyp mice compared with controls, indicating that catabolism of calcitriol is probably increased (12, 85). Interestingly, when Hyp mice are placed on phosphate-restricted diets, their calcitriol concentrations paradoxically decrease (56).

Studies of vitamin D metabolism in the Gy mouse are more controversial. Davidai et al. (14) found that activity of renal 25(OH)D-1alpha -hydroxylase in the Gy mouse was comparable to that of phosphate-depleted mice and much greater than that of normal controls. Of note, serum calcitriol concentrations were higher in Gy mice than controls but not as high as normal mice on phosphate-depleted diets (14). Tenenhouse et al. (82) were unable to detect a statistically significant difference in calcitriol concentrations between Gy and normal mice on control diets. Additionally, when Gy and normal mice were placed on phosphate-restricted diets, normal mice appropriately increased their calcitriol concentration whereas the calcitriol concentration in Gy mice paradoxically dropped substantially below that of Gy mice on the control diet. Phosphate depletion also inappropriately increased 24-hydroxylase activity in Gy mice, which likely contributed to the paradoxical decrease in calcitriol concentrations. Furthermore, 24-hydroxylase activity was elevated in Gy mice on both the control and phosphate-depleted diets, and these investigators concluded that vitamin D metabolism is abnormal in Gy mice.

Since interstrain differences could be responsible for the differences in vitamin D metabolism proposed by Davidai et al. (14), Meyer et al. (59) transferred the Hyp mutation to the B6C3H background. While studying both mutants on the same genetic background, they did not find a difference in calcitriol concentrations between Hyp and Gy mice. However, in both Hyp and Gy mice, calcitriol concentrations were affected by calcium concentrations in the diet. Thus it is possible that much of the observed differences in vitamin D metabolism between Gy and Hyp mice were secondary to differences in background strain and diet.

    POSITIONAL CLONING APPROACH

To gain a better understanding of this disorder, we used the positional cloning approach to locate and clone the HYP gene (38). The advantage of this approach is that knowledge of gene function and tissue expression is not necessary to locate the disease gene. Thus one does not need to make any assumptions about the gene or its function. This approach has been used to identify an ever-expanding number of disease genes (8).

To find a gene using the positional cloning approach, investigators first use linkage analysis to determine the chromosomal location of the disease gene. This method is again used to identify genetic markers that closely flank the disease gene. Once tightly linked flanking markers are determined, a "contig" map is constructed encompassing this region. Contigs are constructed from overlapping pieces of cloned DNA in cosmid, P1, or yeast artificial chromosome (YAC) vectors. Once the region between the flanking markers is covered by the contig, the DNA is analyzed thoroughly to identify all genes present in that region. These genes are tested for mutation in affected individuals.

    LINKAGE STUDIES IN X-LINKED HYPOPHOSPHATEMIC RICKETS

Genetic linkage analyses initially localized the HYP gene to distal Xp (55, 67), and further studies by Thakker et al. (86) established the locus order Xpter-DXS43-HYP-DXS41-Xcen. New genetic markers in Xp22 were required to reduce the candidate region to a smaller size suitable for searching for gene transcripts. Examining five large kindreds, we established DXS257 as the closest flanking marker on the telomeric side, and a second new marker, DXS365, was found to be tightly linked to HYP with no recombination events (21). Rowe et al. (73) also found tight linkage with another new marker, DXS274. The linkage analyses until this point had been performed with restriction fragment length polymorphism (i.e., RFLP) markers (4); however, microsatellite markers were being rapidly established as more powerful tools for genetic linkage analysis (48, 87), since they are generally more polymorphic. Through a combination of examining new families and using microsatellite markers generated from the DXS274 and DXS365 markers, recombination events were detected, which led to an ordering of the genetic markers with respect to HYP as follows: Xpter-DXS43-DXS257-DXS365-HYP-DXS274-DXS41-Xcen (24, 26, 71). Hence, DXS365 and DXS274 were the newly established flanking markers on the telomeric and centromeric sides of the gene, respectively. Construction of a YAC contig in this region (described below) later showed these markers to be separated by a physical distance of ~1-1.5 Mb.

    CONTIG CONSTRUCTION IN THE CANDIDATE REGION

Xp22 markers (DXS365 and DXS274) were used to screen YAC libraries, constructed from human DNA (1, 44). Three YACs were isolated containing the DXS365 locus, and six were isolated containing DXS274. YACs were subsequently selected for generation of end probes, as well as generation of Alu polymerase chain reaction (Alu-PCR) probes, and these were used to rescreen YAC library filters. An end probe from a DXS274-containing YAC and Alu-PCR products from a DXS365-containing YAC identified several YACs in common. Hence, a YAC contig was constructed consisting minimally of three overlapping nonchimeric YACs as follows: a DXS365-containing YAC, one new YAC (not containing either DXS365 or DXS274), and a DXS274-containing YAC (29). The total size of the region was estimated to be 1.5 Mb.

Once the YAC contig was obtained, this was used to isolate new smaller genomic clones [cosmids, P1s, and P1 artificial chromosomes (PACs)] to facilitate the identification of genes in the region. YAC inserts were used to screen gridded cosmid libraries (average insert size 40 kb), a P1 library (average insert size 80 kb), and a PAC library (average insert size 130 kb). These clones were assembled into new contigs by hybridization with end probes, and overlaps were confirmed by restriction digests.

    REDUCING THE SIZE OF THE CANDIDATE REGION

Several cosmid and P1 clones were selected for generation of new microsatellite markers. Southern blots containing these clones were screened with a GT-polymer probe to identify fragments likely to contain a polymorphic CA repeat. Sequences were obtained flanking the CA repeat, and primers were designed to amplify the repeat in HYP families. Two new markers were obtained, DXS1683 (25) and DXS7474 (69), and these were examined in 20 large HYP kindreds. Through this analysis, it was possible to place DXS7474 telomeric of HYP (70) and DXS1683 on the centromeric side (26), and these became the closest flanking markers to the disease. Hence, the candidate region could be narrowed to ~350 kb, which is contained within one of the original YAC clones.

    ISOLATION OF GENE TRANSCRIPTS IN THE CANDIDATE REGION

Three complementary methods were used for gene transcript isolation, i.e., exon trapping (5), cDNA selection (50), and genomic sequencing. In the exon trapping procedure, genomic DNA (e.g., a cosmid clone) is cloned into a new vector that contains functional 5' and 3' splice sites flanking the cloning site. Upon transfection in mammalian cells, splicing can occur if the cloned genomic fragment contains an exon in the correct orientation. Isolation of RNA and reverse transcription (RT)-PCR experiments allow recovery of the spliced exons. This method was performed for several cosmids and P1 clones in the HYP region. cDNA selection is an alternative strategy involving the hybridization of cDNA libraries to genomic clones, to enrich for cDNAs present in the genomic region of interest. A pool of cosmid, P1, and PAC clones that was known to cover the entire HYP candidate region was prepared. Random-primed cDNA libraries were generated from fetal brain, fetal liver, and adult muscle RNA and hybridized to the pool of genomic clones. Those selected cDNAs were subsequently eluted and cloned. The cDNAs were subsequently arrayed on filter membranes and hybridized systematically with genomic fragments from the candidate region and trapped exons. Positively hybridizing cDNAs were rescreened against the cosmid, P1, and PAC clones to confirm their localization. cDNAs from the entire region were then sequenced and compared with the sequences in the public databases.

To screen for deletions in patients, whole cosmids were hybridized to Southern blots of patient DNAs for the detection of aberrant sized bands or absent bands. In total, ~150 HYP patient DNAs were screened in this way. Four deletions were identified in closely situated cosmids (177, 234, 611, and 1005), implying that the gene causing HYP spanned this region (see Fig. 1). Hence, attention was focused on the DNA contained in these four cosmids; we began genomic sequencing this entire region and closely analyzing the localized cDNAs. In parallel, restriction maps were produced from the cosmids across this region, to verify the extent of the deletions and to more finely map the cDNA clones.


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Fig. 1.   Schematic representation of the PEX gene. Predicted protein structure of PEX is at top. PEX consists of 749 amino acids and contains hydrophobic sequences likely to be a transmembrane domain (solid box), 10 cysteine residues (C) in the COOH-terminal domain, which are highly conserved in the neutral endopeptidase family, and two zinc binding motifs, HEXXH and ENXADXGG (hatched box and a slim line, respectively). These motifs contain key residues ("H...H" in the first motif and "E" in the second) required for binding a zinc atom. PEX consists of 22 exons, and these are represented below the PEX protein. Several HYP patients have been identified (TK11, 3941, 4205, and BB39) that have deletions of one or more PEX exons (33). A cosmid contig containing 9 overlapping cosmids was constructed to aid the positional cloning of the PEX gene, and genomic sequence analysis has revealed that PEX spans a genomic region of 220 kb.

    IDENTIFICATION OF THE PEX GENE

The selected cDNA clones had relatively small insert sizes (400-800 bp); however, sequence analysis identified several that were overlapping. In this way it was possible to reconstruct a longer sequence of a gene spanning the patient deletions. Comparison to the sequence databases revealed homologies of this new gene to a family of zinc metalloprotease genes (36), which includes neprilysin (NEP; see Ref. 13), endothelin-converting enzymes (ECE1 and ECE2; see Refs. 28, 74, 90), and the Kell antigen (45). We named this new gene PEX for phosphate-regulating gene with homologies to endopeptidases on the X chromosome (38). By screening PEX cDNAs against the patient DNA panels, it became obvious that each deletion removed at least one PEX exon. Experiments were then performed to complete the PEX gene structure; sequences of other exon trap products and selected cDNAs were examined for evidence of homology to endopeptidases, RT-PCR experiments were performed to link up these sequences, gene prediction programs were used to recognize exons in the genomic sequence, and a 5' rapid amplification of cDNA ends (RACE) reaction was performed in an attempt to identify the 5' end of the gene. PEX is now known to consist of 22 exons spanning 220 kb of genomic sequence (30), which is the majority of the region defined by the microsatellite markers DXS7474 and DXS1683. Interestingly, the DXS1683 marker is situated in the 3' untranslated region of PEX.

The predicted amino acid sequence of PEX suggests that its protein structure closely resembles that proposed for NEP, ECE1 and ECE2, and Kell, which are type II integral membrane proteins. There is evidence for a short NH2-terminal cytoplasmic domain, followed by a hydrophobic region, which is likely to be a transmembrane domain, and then a large extracellular domain. This latter domain contains several highly conserved sequence motifs thought to be involved in zinc binding and substrate catalysis. Specifically, these motifs are <UNL>H</UNL>EXX<UNL>H</UNL> (found in PEX exon 17) and <UNL>E</UNL>NXADXGG (PEX exon 19), in which the underlined residues have been shown in NEP to be zinc binding ligands (46) and the aspartic acid residue has been shown to be involved in substrate catalysis (47). There are in addition 10 cysteine residues throughout the extracellular domain that are likely to be involved in protein folding; these residues are highly conserved in NEP, ECE1 and ECE2, and Kell.

Cloning the human PEX gene has led to a relatively rapid cloning of the mouse Pex cDNA, which has high homology to human PEX (17, 79). Of interest, neither the human nor murine PEX/Pex genes have "classic" Kozak sequences (41). PEX is one of only 3% of known genes that does not have a purine at the -3 position before the ATG initiation sequence (17, 30, 79). This finding may be significant, because, in general, genes that do not have good Kozak sequences tend to be posttranscriptionally regulated (42).

Although little is known about the Kell blood group protein, ECE1, ECE2, and NEP function as ectoenzymes. NEP degrades/inactivates several small peptides including substance P, bradykinin, and enkaphalin. ECE1 and ECE2, on the other hand, convert big endothelin-1 to endothelin-1, the active form (28, 90). Thus it is likely that PEX functions to either activate or degrade a peptide hormone.

    EXPRESSION PATTERN

Although RT-PCR can be used to amplify PEX from lymphocyte and fetal brain RNA (38), it is not likely that these are the physiologically relevant tissues of expression. Database searches (17, 30) identified a highly significant match between the 5' end of PEX and an express sequence tag (EST) containing the 5' end of a rat incisor cDNA clone (GenBank accession no. R47026). Thus PEX is probably expressed in teeth and this may result in the high frequency of tooth abscesses in HYP patients. Du et al. (17) detected a 6.6-kb transcript in mouse bone and mouse osteoblasts with Northern blots using a mouse Pex cDNA. In light of the experiments that demonstrate an osteoblast defect in Hyp mice, it is not surprising that PEX is expressed in the osteoblast. In a more recent study, Beck et al. (3) detected Pex message from mouse calvaria, long bone, and lung and, to a lesser extent, brain, testis, and muscle by RT-PCR. They did not find Pex message in mouse kidney, heart, or liver. These findings in bone and lung were confirmed by ribonuclease (RNase) protection assay, a method that requires greater expression levels than RT-PCR. Of note, levels of expression of Pex are two orders of magnitude less than that of beta -actin (3). They also detected Pex expression on Northern blots of polyA RNA from mouse bone and lung. These investigators also looked for PEX expression in human fetal tissues. They found expression by RT-PCR in calvaria, long bone, lung, ovary, and skeletal muscle (3). RNase protection confirmed expression in calvaria, ovary, lung, and muscle. PEX expression was approximately seven times more abundant in calvaria than in lung, ovary, or muscle (3). Additional data are provided by Grieff et al. (32), who found PEX expression in adult ovary and lung as well as fetal lung and liver. In light of these findings there are several tissues that could play a role in phosphate homeostasis, and PEX may have roles in processes unrelated to phosphate homeostasis.

    PEX MUTATIONS

To establish with certainty that PEX is the HYP gene, we looked for point mutations in HYP patients. In our initial efforts, we detected a frame-shift mutation caused by the loss of a TC dinucleotide in PEX exon 6. We also found two point mutations in the splice acceptor site of the PEX exon 7. These two mutations led to exon skipping (38). More recent efforts (30, 72) have demonstrated mutations in almost every exon, and PEX mutations have been found by other investigators (15, 35). Although mutation detection is still ongoing, there does not appear to be a single common mutation that results in the HYP phenotype. Additionally, all of the mutations described so far appear to be loss of function mutations.

The cloning of the mouse Pex cDNA (17, 79) provides an opportunity to interpret previous studies of Gy and Hyp mice in a new light. Since there are phenotypic differences and an alleged biochemical difference between these mice, several investigators have proposed that there were two closely localized genes that when mutated both resulted in phosphate wasting (14, 58, 76). The occurrence of a single recombination event between Hyp and Gy (54) further supported this conclusion. Strom et al. (79) have, however, determined that the Hyp mouse has a deletion of the last seven Pex exons, and the Gy mouse has a deletion at the 5' end of the gene that involves the first 3 exons and an undetermined amount of upstream sequence. Hence, Hyp and Gy are allelic mutations of Pex. The human PEX gene covers a distance of 220 kb of genomic DNA (30), and it is likely that the mouse Pex gene covers a similar distance. Since the Hyp and Gy mutations occur at opposite ends of the gene, it is not surprising that a recombination event was detected between the two deletions.

Despite the fact that Hyp and Gy both have mutations in Pex, there are still phenotypic differences between the two mutants. One enticing possibility is that the location of the mutations affects the phenotype. However, there does not appear to be a strong genotype/phenotype correlation in humans (unpublished observations). It has also been suggested that differences in the biochemical manifestations of the mutations may be related to background strain and dietary differences. Of note, when Hyp mice are bred onto the B6C3H background (the background on which the Gy mouse is bred) some of the mice exhibit circling behavior (58). However, the Gy male does not survive on the C57BL/J6 background (58). An alternative possibility is that one or both of the deletions may involve another gene in the region that influences the overall phenotype. Further work is required to provide an adequate explanation. In any event, the identification of the Hyp and Gy mutations provides strong evidence that Pex is the only phosphate-regulating gene on this portion of the X chromosome.

    POSSIBLE ROLES FOR PEX IN NORMAL PHOSPHATE HOMEOSTASIS

Despite that fact that mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets, its role in the pathophysiology of HYP is not immediately obvious. Several observations should be taken into account when considering possible mechanisms. First, HYP is an X-linked dominant disorder with little, if any, gene dosage effect. In this regard, it is possible that mutations in the PEX gene result in a dominant negative effect. Alternatively, HYP could be a haploinsufficiency disorder where having one-half the normal amount of PEX gene in females (or null amounts in males) could result in the disease phenotype. This latter possibility is favored, because the TK11 patient deletion (Fig. 1) and the murine Gy mutation almost certainly result in lack of message production (38, 79), hence ruling out the possibility of a dominant negative effect. Second, as noted above, the Hyp and Gy mouse mutations result in decreased levels of Npt-2, the high-affinity/low-capacity sodium-dependent phosphate cotransporter. Thus it is likely that PEX serves to directly or indirectly regulate the expression of this transporter. Third, studies demonstrating PEX expression in osteoblasts and those studies that demonstrate an osteoblast defect in the Hyp mouse indicate that the osteoblast defect is responsible for at least part of the mineralization abnormalities that are seen in the disease. These studies also indicate that the osteoblast could play an important role in regulating phosphate homeostasis, although there is currently insufficient data to fully support this contention. Fourth, the existence of tumors that secrete phosphatonin, as well as parabiosis data in the Hyp mouse, support the notion that the pathophysiology of the disease involves the elaboration of a humoral phosphate-wasting factor. Since PEX mutations, which result in the disease phenotype, are loss-of-function mutations and not activating mutations, it is clear that PEX is not phosphatonin. However, the PEX gene product may play a role in regulating the concentration of phosphatonin.

There are several possible roles for the normal PEX protein in phosphate homeostasis. Since PEX is a member of the neutral endopeptidase family, it is possible that PEX degrades/inactivates phosphatonin. Thus mutations in PEX could interfere with this process and result in excessive concentrations of phosphatonin. However, if this is the case, then one might predict that parabiosis between a Hyp and normal mouse would rescue the Hyp phenotype (i.e., the normal Pex protein might be expected to degrade excessive phosphatonin from the mutant animal). However, parabiosis did not rescue the Hyp phenotype. Instead, normal mice, when parabiosed to Hyp mice, started to waste phosphate (57). Although resolution of this apparent discrepancy awaits additional data, it is possible that the kidney is exposed to the high phosphatonin level before the PEX protein has a chance to degrade it. Alternatively, the normal animal may not adequately upregulate PEX to degrade the excessive amounts of phosphatonin generated by the mutant animal. Another potential mechanism of action that is in keeping with an enzymatic role for PEX is that, under normal circumstances, PEX could function to activate a phosphate-conserving hormone. This possibility has become more plausible in light of recent data that human stanniocalcin stimulates phosphate reabsorption when administered to rats (64). However, this model would also predict that parabiosis of normal mouse to Hyp mouse would rescue the Hyp phenotype. An additional possibility that fits in with the currently available data is that the PEX gene indirectly functions to inhibit the expression of phosphatonin. Thus mutations of the PEX gene would result in overexpression of phosphatonin and lead to renal phosphate wasting.

    CONCLUSION
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Abstract
Introduction
Conclusion
References

As is frequently the case when disease genes are identified by the positional cloning approach, identification of the gene does not guarantee immediate understanding of the pathophysiology. However, until the PEX gene was cloned, no one had ever proposed that an enzyme played a role in the disease. Cloning the PEX gene has provided investigators with new opportunities to understand the pathophysiology of the disease and normal phosphate homeostasis. Future research aimed at identifying the PEX substrate, PEX tissue expression, and PEX regulation will undoubtedly add to our understanding of the disease. A more complete understanding of the pathogenesis of HYP and other disorders of phosphate wasting will provide vital clues to understanding normal phosphate homeostasis. The available data indicate that control of phosphate homeostasis is a complex but fascinating process.

    ACKNOWLEDGEMENTS

Work performed in our laboratories was supported by National Institutes of Health Grants AR-42228, AR-27032, and MO1-RR-30, as well as Grant 5-P60-AG-11268 from the Claude Pepper Older Americans Independence Center, and by grants from the Commission of the European Communities and from the Peter und Traudl Engelhorn Stiftung.

    FOOTNOTES

Address for reprint requests: M. J. Econs, Dept. of Medicine, Indiana University Medical Center, 975 W. Walnut St., IB 445, Indianapolis, IN 46202.

    REFERENCES
Top
Abstract
Introduction
Conclusion
References

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