Evidence for Phex haploinsufficiency in murine X-linked hypophosphatemia

Abstract

Mutations in the PHEX gene (phosphate-regulating gene with homology to endopeptidases on the X-chromosome) are responsible for X-linked hypophosphatemia (HYP). We previously reported the full-length coding sequence of murine Phex cDNA and provided evidence of Phex expression in bone and tooth. Here, we report the cloning of the entire 3.5-kb 3'UTR of the Phex gene, yielding a total of 6248 bp for the Phex transcript. Southern blot and RT-PCR analyses revealed that the 3' end of the coding sequence and the 3'UTR of the Phex gene, spanning exons 16 to 22, are deleted in Hyp, the mouse model for HYP. Northern blot analysis of bone revealed lack of expression of stable Phex mRNA from the mutant allele and expression of Phex transcripts from the wild-type allele in Hyp heterozygous females. Expression of the Phex protein in heterozygotes was confirmed by Western analysis with antibodies raised against a COOH-terminal peptide of the mouse Phex protein. Taken together, these results indicate that the dominant pattern of Hyp inheritance in mice is due to Phex haploinsufficiency.

The Wrickkened Pathways of FGF23, MEPE and PHEX

“…Moreover, multiple new mutations arise each generation such that haploinsufficiency at most loci would, in fact, be incompatible with life itself. Therefore, the finding that the Mepe null-mutant (Mepe (-/-) and the Hyp mouse (Phex -/-) both exhibit haploinsufficiency is truly remarkable and in itself compelling evidence of a MEPE-PHEX association, regardless of the other phenotypic links described earlier (Qiu et al, 1993(Qiu et al, , 2003Wang et al, 1999;Gowen et al, 2003). Moreover, the fact that X-linked hypophosphatemic rickets is a dominant disease is perplexing, and the discovery that PHEX function (a putative protease) is independent of gene dose is fascinating.…”

“…The ASARM model, however, is consistent with these findings. In heterozygous Hyp females, both the normal and abnormal Phex alleles are expressed (Wang et al, 1999). Thus, in Hyp female heterozygotes, there is a chimeric population of normal and abnormal osteoblasts (lyonization or X-chromosome inactivation; Lyon, 1988), with commensurate excess of localized bone production of ASARM peptide.…”

The X chromosome deletion in <i>Hyp</i> mice extends into the intergenic region but does not include the <i>Sat</i> gene downstream from <i>Phex</i>

“…Thus, to determine Phex intronic sequences and intergenic sequences downstream from Phex, we isolated a BAC clone containing the region of interest. A mouse BAC library was screened with a Phex cDNA probe spanning exons 16 to 19 in the 3) region of the gene that is deleted in the Hyp mouse (Beck et al, 1997;Strom et al, 1997;Wang et al, 1999). We obtained five positive BAC clones (249O21, 338J7, 379H17, 418M20, and 476D24) which were characterized by PCR using primers in the 3) end of the coding region and 3) Fig.…”

“…The 5) boundary of the deletion occurs somewhere in intron 15 Wang et al, 1999), which is F27 kb in length (http://www.ensembl.org). In addition, it was demonstrated that the 3.5-kb 3) untranslated region (UTR), which is part of exon 22, is deleted in Hyp mice (Wang et al, 1999).…”

“…The 5) boundary of the deletion occurs somewhere in intron 15 Wang et al, 1999), which is F27 kb in length (http://www.ensembl.org). In addition, it was demonstrated that the 3.5-kb 3) untranslated region (UTR), which is part of exon 22, is deleted in Hyp mice (Wang et al, 1999). Although the size of the Hyp deletion was initially estimated to be between 18 and 33 kb (Beck et al, 1997), more precise information about the extent of the deletion is necessary, particularly with regard to the status of the downstream gene, spermidine/spermine N1-acetyltransferase (Sat; formerly Ssat).…”