Three novel mutations in the PHEX gene in Chinese subjects with hypophosphatemic rickets extends genotypic variability

Abstract

Mutations in the phosphate-regulating endopeptidase homolog, X-linked, gene (PHEX), which encodes a zinc-dependent endopeptidase that is involved in bone mineralization and renal phosphate reabsorption, cause the most common form of hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH). The distribution of PHEX mutations is extensive, but few mutations have been identified in Chinese with XLH. We extracted genomic DNA and total RNA from leukocytes obtained from nine unrelated Chinese subjects (three males and six females, age range 11-36 years) who were living in Taiwan. The PHEX gene was amplified from DNA by PCR, and the amplicons were directly sequenced. Expression studies were performed by reverse-transcription PCR of leukocyte RNA. Serum levels of FGF23 were significantly greater in the patients than in normal subjects (mean 69.4 ± 18.8 vs. 27.2 ± 8.4 pg/mL, P < 0.005), and eight of the nine patients had elevated levels of FGF23. Germline mutations in the PHEX gene were identified in five of 9 patients, including novel c.1843 delA, donor splice site mutations c.663+2delT and c.1899+2T>A, and two previously reported missense mutations, p.C733Y and p.G579R. These data extend the spectrum of mutations in the PHEX gene in Han Chinese and confirm variability for XLH in Taiwan.

Fig. 1

Molecular analysis of PHEX. a Proband 1 had a c.1843 delA mutation in his copy of the PHEX gene. b Proband 2 was heterozygous for c.663+2delT, resulting in alternative splicing of mRNA from this allele. The amplicons generated by RT-PCR of RNA from a normal subject and the patient are shown and indicate that the patient had a smaller band (694 bp) than expected (771 bp) for the wild-type allele. The RT-PCR product of this patient was sequenced, and the 5′-splice site of intron 5 was shifted 77 nucleotides upstream in exon 5. c Patient 3 was hemizygous for the c.1899+2T>A mutation that resulted in an abnormally spliced mRNA in which the 5′-donor splice site of intron 18 was shifted 131 nucleotides upstream of the native donor splice site, resulting in skipping of exon 18. The amplicon corresponding to wild-type cDNA is 521 bp, and the corresponding mutant amplicon is 390 bp. d Patient 4 had a C733Y missense mutation (TGT → TAT) in codon 733 of exon 22. e Patient 5 had a G579R missense mutation (GGA → AGA) in codon 579 of exon 17

Fig. 2

The distribution of PHEX gene mutations among Chinese with XLH is shown. Mutations in exon 18, in which the zinc-binding domain is located, are the most common and account for 30% (4/13) of PHEX mutations in Chinese subjects