The role of the PHEX gene (PEX) in families with X-linked hypophosphataemic rickets.

For over a hundred years, the bane of rickets (a disease of bone), has been prominent in those countries that have participated in, and seeded, the industrial revolution. Industrialisation had major effects of the demography of populations, and many people moved to dark, heavily industrialised cities to find work. It soon became apparent that rickets could be cured by supplementing the diet with cod liver oil and exposure to sunlight. This in turn led to the discovery that photoactivation of 7-dehydrocholesterol was required to produce vitamin D, an indispensable regulator of bone mineral metabolism. Although inadequate exposure to light and poor dietary intake are the main causes of rickets and osteomalacia, recent research has confirmed the role of familial, and tumour forms of the disease. This review will describe the recent advances in our knowledge of the molecular defects in X-linked hypophosphataemic rickets (HYP), and oncogenic hypophosphataemic osteomalacia (OHO). Although HYP and OHO have different primary defects, both diseases have similarities that suggest a linked or overlapping pathophysiology. Also, without doubt, the recent cloning of the gene defective in HYP (the PHEX gene), has given researchers a new reagent to explore the molecular regulation of bone and its links to kidney endocrine function. The fact that the PHEX gene codes for a Zn metallopeptidase raises new and intriguing questions, and adds new momentum to the research on diseases of bone mineral metabolism.

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP).

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.

Candidate 56 and 58 kDa protein(s) responsible for mediating the renal defects in oncogenic hypophosphatemic osteomalacia.

The effects of tumor-conditioned media (TCM) derived from cultured cells from an oncogenic hypophosphatemic osteomalacia (OHO) tumor on transformed human kidney cells were investigated. Dose-dependent cell detachment and aggregation occurred in kidney cells cultured in serum-free medium supplemented with TCM, but not in skin fibroblast controls, or in kidney cells cultured in the presence of serum. Kidney cells exposed to TCM in the presence of serum (0.5%) had reduced Na(+)-dependent phosphate cotransport (36%, p < 0.04) and increased 1alpha-hydroxylase activity (48%, p < 0.05). In contrast, TCM had no significant effect on Na(+)-dependent alpha-methyl-glucose transport. To investigate these effects further, serum from an OHO patient, before and after tumor resection, was used to raise polyclonal antiserum to tumor-derived products (preoperative and postoperative antiserum, respectively). Changes in Na(+)-dependent phosphate cotransport and vitamin D metabolism induced by TCM were prevented by the addition of preoperative but not postoperative antisera. Furthermore, Western analysis revealed the presence of two proteins (56-58 kDa) in TCM media screened with preoperative antisera. These proteins were not detected by postoperative antisera and were absent in skin fibroblast control media. Direct inhibition of Na(+)-dependent phosphate cotransport by phosphonoformic acid did not affect 1,25-dihydroxy vitamin D(3) synthesis. These studies provide support for a circulating component affecting phosphate handling and vitamin D metabolism in OHO.

Cell wall characteristics of Pseudomonas aeruginosa and its carbenicillin-induced L-form.

L-forms of Pseudomonas aeruginosa were induced and cultured on a medium supplemented with carbenicillin. Morphological studies of the passaged variant revealed the presence of a triple-layered cell wall similar to that found in the parent species. Furthermore, the L-form was found to be more susceptible to gentamicin, kanamycin, tetracycline and colistin sulphate. Chemical analysis of the lipopolysaccharide fraction showed a difference in phosphorus content, and changes in cell wall envelope fatty acid content were also exhibited. It is suggested that these differences may influence the transport of certain antibiotics through the cell wall.