Molecular Diagnosis Of Hypophosphatemic Rickets And Functional Research Of Novel Mutations In PHEX Gene In Vitro

Background and Objectives:In human body,phosphorus functions in multiple vital movements by varies ways within a homeostasis.When the homeostasis breaks,the physiological function will be affected,and thus disease happens.Hypophosphatemic rickets(HR),characterized by hypophosphatemia and abnormalities in bone development,is the most representative type in the abnormal diseases of phosphorus metabolism.It involves a series of metabolic bone disease that is causative of genetic factors.Among them,X-linked dominant hypophosphatemic rickets(XLHR)occurred the most,and has dealt numerous patients a heavy blow,accompanied their families and the whole society under the long-term blight of high teratogenicity and disability rate.Clinically,patients with XLHR usually have the same symptoms compared with the other types of HR diseases.Therefore,a golden standard to identify these diseases is to give gene analyses.Current researches have proven that the gene PHEX is the major disease-causing gene for XLHR.Patients with loss-of-function mutations in PHEX will receive less phosphorus reabsorption in kidney and in small intestine,which in turn lead to hypophosphatemia as well as impaired bone mineralization.In order to increase the detection rate of gene mutations,we,in this study,performed multiple sequencing methods upon the patients who were diagnosed with HR by clinic as well as their families.Meanwhile,we firstly confirmed the pathogenesis in the mutant sites only detected in Chinese patients by functional analysis in experiments,which not only provided a strong evidence on the diagnose of XLHR patients,but also broadened the mutation spectrum and simultaneously gave a good reference value for the analysis of genotype-phenotype relationship and pathogenesis as well.Methods:1.Make a collection on the proband diagnosed with HR as well as their families,and analysis the clinical features upon them;2.Peripheral blood of patients and their family members was collected for DNA extraction.Using Sanger sequencing,along with the multiplex ligation-dependent probe amplification and whole exome sequencing,we made the mutation analyses on each affected family.3.After identifying the relevant mutation sites,we performed the analyses of the pathogenesis on some novel(de novo)PHEX mutations by bioinformatics analysis and functional experiments in vitro.Results:There were totally 32 patients who had been diagnosis as HR in clinic enrolled in our research.In the analyses of clinical features,all most all the patients harboring the characteristic signs of HR except one only detected with growth retardation.After divided these probands into three groups by the different age of onset,we detected that the age of onset was negatively correlated with the rate of short stature.Next,by using gene sequencing,there were 27 patients detected with the PHEX mutations,and among them,fifteen were novel(de novo)mutations.Besides,the other five patients were found to have mutations in other rickets related genes,such as FGF23,ENPP1,OCRL and SLC34A3.By predictive analytics of bioinformatic analysis,we speculated that all those novel(de novo)mutations would do harm to the structure and functions of proteins and were more prone to be pathogenic.Whereafter,five PHEX novel(de novo)mutations were selected to identify the pathogenicity via vitro experiments,and found all of them presented distinct abnormalities compared with the wild type PHEX,like the productions of the truncated proteins,an impaired glycosylation and a difference in protein expression level.By analysis the mutant enzyme activity in three of the five,we also detected that these mutations have impaired the enzyme activity.Finally,combined with clinical and bioinformatics analysis,we considered those three novel(de novo)mutations in PHEX do have strong pathogenic potential.Conclusion:1.Among all the enrolled HR patients,the age of onset was negatively correlated with the rate of short stature.2.The combination of multiple sequencing methods could compensate for the limitations of sequencing when applied alone.3.All the mutations detected in this study were considered to be pathogenic.4.Bioinformatics analysis combined with the functional experiments,to some extent,could explain the differences of clinical phenotypes.