Molecular Genetics Of X-linked Hypophosphatemic Rickets In Children

Background: Hypophosphatemic rickets(HR)is a genetic disorder characterized by insufficient reabsorption of phosphate(Pi)in the proximal renal tubule and inappropriately low or normal serum 1,25-dihydroxyvitamin D3 levels,which cause growth retardation,rickets and osteomalacia.The most frequent form of clinical HR is X-linked hypophosphatemic rickets(XLHR;MIM#307800),which accounts for approximately 80% of familial cases of hypophosphatemia.Pathogenic mutations in the phosphate regulating endopeptidase homolog X-linked(PHEX;MIM#300550)gene have been identified as the main cause of this form of HR.In this study,we investigated the molecular bases and genotype-phenotype correlations of 53 pediatric patients with XLHR.Methods: Clinical and molecular data were collected from 53 patients with variants in PHEX.All patients were analyzed for the PHEX gene by direct sequencing or next-generation sequencing.When no mutations were detected in PHEX gene,multiplex ligation-dependent probe amplification(MLPA)analysis was performed.The effect of splicing mutations was investigated by a minigene splicing assay using the p SPL3 plasmids.To conduct a genotype-phenotype correlation analysis,genotypes were subdivided into two groups: truncating mutations and non-truncating mutations groups.Results: The 53 XLHR children comprised 24 familial cases and 29 sporadic cases.The male-to-female ratio of the 53 patients was 31:22.Median age at diagnosis was 28 months.Overall,47 different PHEX mutations were identified,including 15 frameshift,seven nonsense,seven splicing alteration,12 missense,one in-frame deletion and five exonic deletions.27 of the 47 variants were not previously described in the literature or entered in the HGMD.The minigene splicing assay confirmed that IVS6+1G>C,IVS6+1G>A,IVS9+1G>A,IVS11+1G>A and IVS20+2T>G caused exon skipping.The IVS15+1G>A substitution at position +1 crippled the wild-type donor splice site by activating an aberrant new donor site,which led to the inclusion of 74 bp from intron 1).Notably,the IVS5-1G>C splice accept site mutation resulted in an incomplete loss of exon 6.The severity of bone phenotype,height SDS,serum Pi,TRP and alkaline phosphatase levels at diagnosis did not show an evident correlation between the truncating and non-truncating mutation types in our cohort.Conclusion: 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.Background: X-linked hypophosphatemia(XLHR)is caused by loss-of-function mutations in the PHEX gene.Considerable controversy exists regarding genotype-phenotype correlations in XLHR.In this study,we investigated the molecular bases and genotype-phenotype correlations of 53 pediatric patients with XLHR.To further delineate the characteristics of PHEX variants underlying the genotype-phenotype trend,we functional characterized 10 PHEX gene variants in vitro.Methods: Seven PHEX non-truncating mutation and three truncated mutations were characterized in vitro by assessing their effects on PHEX expression,subcellular localization,glycosylation pattern and endopeptidase activity.Moreover,the effect of the two molecular chaperone drugs(4-PBA and VX809)on the expression and localization of the PHEX mutants was investigated by western blot and immunofluorescence.Results: Functional assays 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.Cys77 Tyr,p.Cys85 Ser,p.Ile281 Lys,p.Ile333 del,p.Ala514 Pro and p.Gly572 Ser mutants were not secreted into the medium and remained trapped inside cells in an immature form,while the p.Gly553 Glu mutant was terminally glycosylated and secreted into the medium.We further assessed the endopeptidase activity of the p.Gly553 Glu mutant using a quenched fluorogenic peptide substrate and revealed that the activity of p.Gly553 Glu significantly reduced to 13% compared with wild-type.Treatments of 4-PBA and VX809 failed to rescue the decreased expression level and localization defect of the PHEX mutants.Conclusion: This is the largest genetic report of a case series pediatric patients with XLHR;concurrently,it is the first study of a comprehensive functional characterization of PHEX gene variants identified in XLHR patients.The results provide a similar experimental portrait for the truncating and non-truncating variants in PHEX.These data not only support the clinical results showing no correlation between disease severity and the type of PHEX mutation but also provide novel insights into therapeutic strategies.