Functional Characterization of PHEX Gene Variants in Children With X-Linked Hypophosphatemic Rickets Shows No Evidence of Genotype-Phenotype Correlation

X-linked hypophosphatemia (XLHR) is caused by loss-of-function mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Considerable controversy exists regarding genotype-phenotype correlations in XLHR. The present study describes the clinical features and molecular genetic bases of 53 pediatric patients with XLHR. Overall, 47 different mutations were identified, of which 27 were not previously described in the literature or entered in the Human Gene Mutation Database (HGMD). A high prevalence (72.34%) of truncating variants was observed in XLHR patients. The clinical presentation and severity of XLHR did not show an evident correlation between the truncating and non-truncating mutation types in our cohort. To further delineate the characteristics of PHEX variants underlying this nonsignificant trend, we assessed the effects of 10 PHEX variants on protein expression, cellular trafficking, and endopeptidase activity. Our results showed that the nonsense mutations p.Arg567*, p.Gln714*, and p.Arg747* caused a reduction of protein molecular weight and a trafficking defect. Among seven non-truncating mutations, the p.Cys77Tyr, p.Cys85Ser, p.Ile281Lys, p.Ile333del, p.Ala514Pro, and p.Gly572Ser mutants were not secreted into the medium and remained trapped inside cells in an immature form, whereas the p.Gly553Glu mutant was terminally glycosylated and secreted into the medium. We further assessed the endopeptidase activity of the p.Gly553Glu mutant using a quenched fluorogenic peptide substrate and revealed that the activity of p.Gly553Glu significantly reduced to 13% compared with the wild type, which indicated disruption of catalytic function. These data not only support the clinical results showing no correlation between disease severity and the type of PHEX mutation but also provide helpful molecular insights into the pathogenesis of XLHR. (c) 2020 American Society for Bone and Mineral Research.