High Throughput Functional Verification Of PRPS1 Variations And Genetic Heterogeneity In A Family With Hearing Loss

Part one: Functional validation and pathogenicity mechanism of PRPS1 variationsDeafness is common sensory defect in clinic.According to the statistics of the World Health Organization,more than 5% of the world's population-466 million people suffer from varying degrees of hearing loss,and about 60% of hearing loss are caused by genetic defects.At present,International hereditary hearing loss website?hereditaryhearingloss.org?contains 120 hereditary nonsyndromic deafness genes,48 common apparent deafness genes,76 common occult deafness genes and 5 Xlinked genetic deafness genes.PRPS1 is located in Xq22.3 and encodes phosphate ribose pyrophosphate synthase I?PRS-1 enzyme?,which catalyzes ribose 5-phosphate and ATP to produce AMP and PRPP?5-phosphate ribose-1-pyrophosphate?,PRPP is an important substrate for de novo and remedial synthesis of purine,pyrimidine and pyridine nucleotides in vivo.The increase and decrease of enzyme activity caused by PRPS1 mutation may involve systemic multi-organ and multisystem lesions including deafness.The decrease of enzyme activity of PRPS1 mutants can lead to DFNX1 nonsyndromic deafness,and can also lead to more severe symptoms of Charcot-Marie-Tooth Neuropathy X Type 5 syndrome and Art's syndrome.The increase of enzyme activity of PRPS1 mutants can lead to mild PRS superactivity and severe PRS superactivity.A total of five male patients with hearing loss and one normal control were detected carrying PRPS1 missense mutations in our study.The family members of these patients with deafness were investigated by questionnaire,audiological examination and physical examination.CT,MRI and other clinical data as well as blood samples were collected,and DNA was extracted for MPS sequencing analysis.By collecting blood samples from patients,family members and normal controls outside the family,hemoglobin was extracted and the activity of PRS-1 enzyme was detected in vitro to provide evidence of pathogenicity for the variations.The validation of the pathogenicity of PRPS1 variations includes: 1.Eighteen variations and wild-type PRPS1 expression plasmids such as D65 N,M115T,Q133 P,G174R,N144 S,I292V,etc.,were constructed by pEGFP-N1-Flag eukaryotic plasmid.Representative disease mutant plasmids were selected and transfected into HEK293 FT cells,and the protein was extracted for Western Blot.The results showed that there was no significant difference in the expression of PRPS1 mutant protein among different disease phenotypes.2.Wild-type and six variations PRPS1 plasmids?D65N?M115T?Q133P?G174R?N144S?I292V?were transfected into HEK293 FT cells for immunofluorescence localization.It was found that PRPS1 wild type protein and mutant proteins with different disease phenotypes were concentrated in the cytoplasmic matrix,and there was no significant difference in expression site.3.With the prokaryotic expression system of pET-28 a plasmid and Rosetta strain and HPLC technology,a high-throughput in vitro PRPS1 enzyme activity detection platform was established to quickly identify the pathogenicity of PRPS1 mutation and provide pathogenicity evidence.PRPS1 wild type and 20 mutant plasmids were constructed using pET-28 a His tagged prokaryotic plasmid,and the proteins were expressed in Rosetta host bacteria.PRS-1 enzyme protein was purified by His tag Ni column affinity chromatography.The reaction environment of PRS-1 enzyme was simulated in vitro,and the content of product AMP was detected by high performance liquid chromatography?HPLC?.The difference of PRS-1 enzyme activity among different disease phenotypes was identified.The results showed that the more serious the disease phenotype caused by different mutants of PRPS1,the lower the residual activity of PRS-1.The detection platform was used to study the two new PRPS1 variants identified in this study.The results showed that the activity of Met73 Val mutant protein was lower than that of PRPS1 wild type protein,significantly higher than that of CMTX5 and Art,s syndrome,and the residual enzyme activity was 85.4%.The activity of Ile292 Val mutant protein was similar to that of PRPS1 wild type protein,and the residual enzyme activity was 106.9%.The activities of the two new variants of PRPS1 were consistent with the phenotype of the disease and interpretation of genetic analysis,which also verified the reliability of the high-throughput functional varification platform.4.The purified PRS-1 enzyme protein was subjected to isothermal titration calorimetry?ITC?reaction.The PRS-1 protein was titrated by ATP,and the binding curve was fitted by software.The results showed that the binding ability of PRPS1 mutant?D65N?P276S?K280E?proteins to ATP was lower than that of wild type.In conclusion,through genetic analysis and functional verification,five novel PRPS1 pathogenic mutations and one PRPS1 benign variation were identified in Chinese deaf and control population,which provided evidences for clinical genetic diagnosis and genetic counselling of five families with deafness.Seven novel variants with clear classification were added to PRPS1 mutation database.A high-throughput in vitro PRPS1 enzyme activity assay was developed to establish pathogenicity interpretation and pathogenicity mechanism of PRPS1 variations.Part two: Genetic heterogeneity in a family with hearing lossIn this study,the clinical phenotype and pathogenic genes of a four generation family with hereditary deafness were analyzed.A questionnaire survey,audiological examination,visual system examination and physical examination were carried out among the members of HL-258 family.It was found that the hearing phenotypes of the deaf patients in the family were significantly different and there were two genetic patterns.Two pathogenic mutations,POU4F3:NM₀02700.3: c.976A>G?p.Arg326Gly?and PDZD7: NM₀24895.4: c.490C>T?p.Arg164Trp?were identified by TGE+MPS and bioinformatics analysis in this family.Sanger sequencing confirmed that the mutations were cosegregated with the hearing loss in this family.Conservation analysis showed that the amino acid residues of the mutations were highly conserved in different species.The three dimensional protein structure showed that the mutations disturbed the stability of the local secondary structure to some extent and affected the protein subcellular localization and local hydrophobicity.Mutations on POU4F3 and PDZD7 were identified in a four-generation family with hereditary hearing loss,which were transmitted in autosomal dominant inheritance and autosomal recessive inheritance respectively.This family enlightens us that there may be multiple genes in one family with family history of deafness.When we find the pathogenicity mutation of a gene,the genetic diagnosis of deafness may not be finished.It is suggested that high-throughput detection technology platform should be applied for clinical gene diagnosis of deafness to avoid missed diagnosis.