Mutant ATP7B Gene Transcriptional Level Analysis And Its Mutant Protein Three-dimensional Structure Study

Background:Hepatolenticular degeneration (HLD), also called Wilson disease(WD)（,Wilson’s disease,WD）,is an autosomal recessively inherited disorder of copper metabolism.The worldwideincidence rate of1/100000~/30000,Pathogenic gene carriers is about1/90;The main clinicalmanifestations is lower serum ceruloplasmin,Cirrhosis,chronic progressive extrapyramidalsymptoms, Kayser-Fleischer (KF) rings,Psychiatric symptoms, etc. At present, there arehave found a large number of APT7B mutations worldwide. But the study of these mututionson Transcriptional level，and its mutant protein three-dimensional structure is still less, therehave no relevant reports in the domestic. We have previously used the DHPLC screening ofATP7B gene mutations in75WD patients in southern China, Screened a total of38kinds ofmutations, in which10kinds of new mutations reported for the first time at home andabroad.In this study, ATP7B gene DNA mutant sites was amplified by PCR and mRNAmutant sites was amplified by nested PCR (nested polymerase chain nreaction) to analysistranscriptional level of WD patients.Through the online prediction software:ProtParam,Protscale,Mensat3,Analysis of mutant and wild-type ATP7B protein physical andchemical properties,Hydrophobic nature,The structure of the transmembrane region.UsingOnline Swiss-Model software for homology modeling of wild-type ATP7B core region and itsTransmembrane helix，and use this template to study mutant effect on three-dimensionalstructure of the ATP7B protein.Objective:1．Verified that the ATP7B mutant sites is transcribed from DNA to its mRNA.2．Studied on the ATP7B mutant protein Physical and chemical characteristics,hydrophobic nature and transmembrane helix structure.3．Obtained the Three-dimensional structure of the ATP7B protein core region and thetransmembrane helix,and modeling mutant ATP7B protein three-dimensional structure bymodel of wide-type ATP7B Three-dimensional structure, analysis the mutant effect on theATP7B protein structure and function.4．To study the mRNA Transcription of the splice site mutation:1543+1G>T,3244-2A>C.5．Analysis the genotype and phenotype relationship between3244-2A>C,2333G> Theterozygous mutation with2333G> T homozygous mutation patients,to Explore thegenetic significance of the two different mutations.Methods:1．Extracted total DNA,RNA from peripheral blood lymphocytes.2．Using conventional PCR and nested PCR to study on Transcriptional level of mutantATP7B gene(Missense,frameshift and splicing mutations)，Verified ATP7B mutant sites istranscribed from DNA to mRNA.3．Through the online protein analysis software research the physicochemical, hydrophobicproperties and transmembrane helix of the mutant ATP7B protein.4．Using Online homology modeling software to get the core region and the transmembranehelix three-dimensional structure of the ATP7B protein,And use the constructedthree-dimensional structural model of ATP7B protein to model and analysis of the mutanteffect on ATP7B Protein structure and function.5．Using nested PCR Analysis the aberrant transcripts of the splice site mutation.6．Collecting the clinical data and results of genetic testing of3244-2A> C,2333G> Theterozygous mutation and2333G> T homozygous mutation patients to study the genotypeand clinical phenotype relationship between them.Results:1．This study tested the previous screening of10new found or common mutations: sevenmissense mutations2333G>T,2620G>C,2761A>C,2755C>G,3443T>C,3446G>A,3682A>T,an insertion mutation1875-1876insTTAA, two splice site mutation1543+1G>T,3244-2A>C.2. This study demonstrated that Six Missense mutations （2333G>T,2620G>C,2761A>C, 3443T>C,3446G>A,3682A>T）transcript from DNA into mRNA;One splice site3244-2A>Clead to abnormal transcription at the mRNA level; One splice site mutation1543+1G>T is notfound splicing abnormalities;An insertion mutation1875-1876insAATT and a missensemutation2755C>G were not found in the mRNA level.3．Using Online protein physical and chemical properties analysis software ProtParam toanalysis physical and chemical properties of mutant ATP7B protein,the result show thatthere are varying degrees of physical and chemical properties change among mutant ATP7Bprotein.4．Using Protein hydrophobicity analysis software Protscale to analysis hydrophobicity ofthe mutant ATP7B protein:Arg778Leu, Ala874Ser, Ser921Arg, Ile1148Thr,Gly1149Glu,found the Arg778Leu mutant protein amino acid sequence and its adjacent sequenceshydrophobicity Increase,but other four mutant ATP7B protein decrease.5．Though online transmembrane structural analysis software Memsat3found that six pointmutations (Arg778Leu, Ala874Ser,Ser921Arg,Ile1148Thr,Gly1149Glu,Arg1228stop), andone truncated mutation3244-2A>C have an impact on the transmembrane helix. In additionto Arg1228stop mutation,Five missense mutant ATP7B protein have the same change at thelocation of6transmembrane helix.6． Swiss-Model homology modeling analysis show that the mutations affect thethree-dimensional structure of the mutant proteins;Missense mutations in this study:Arg778Leu, Ala874Ser, Ser921Arg, Ile1148Thr, Gly1149Glu resulting three-dimensionalstructure of the ATP7B protein change significantly By prediction:The second and sixthtransmembrane helix structure extended by one residue,the length of two folded structure inthe phosphorylational domain and a folded structure in the transductional domain extended byone residue.Other part of the Mutant protein three-dimensional structure compared withwild-type ATP7B,Slight changes in the spatial location.7．Through one cases of3244-2A> C,2333G>T heterozygous mutation patient Compare withthe2333G> T homozygous mutation patients with genotype,Clinical performance,indicatedthat3244-2A> C mutation is a mutation of serious prognosis. Conclusion:1．Extracted total DNA,RNA from peripheral blood lymphocytes,can do study transcriptionallevel of ATP7B accurately and quickly.2．Using online software ProtParam to analysis the physical and chemical properties of mutantATP7B protein,showed that the physical and chemical properties of the Truncated mutantATP7B protein more change than the missense mutations. Prompted the truncated mutationsmore severely affect the function of the of ATP7B protein.3．Through analysis by hydrophobicity analysis software Protscale,the results showed that thehydrophobicity of the Arg778Leu mutant protein increased significantly, the hydrophobicity ofAla874Ser,Ser921Arg,Ile1148Thr and Gly1149Glu mutant protein declines dramatically;Asthe hydrophobicity of the amino acid sequence is closely related to transmembrane helixformation,Protscale analysis results suggest that the hydrophobicity change may have animpact on transmembrane helix.4．The results of Memsat3transmembrane structural analysis software showed that:Each(Arg778Leu, Ala874Ser,Ser921Arg,Ile1148Thr,Gly1149Glu) missense mutant protein changesome location of transmembrane helix,sixth transmembrane helix transmembrane positionchange in all These mutant proteins. Prompted that the change hydrophobicity of the mutantproteins may be the reason for the change of transmembrane helix location.5．Using homology modeling software Swiss-Model,modeling the wild-type ATP7B proteinthree-dimensional structure Successfully.6． Building mutant protein (Arg778Leu, Ala874Ser,Ser921Arg,Ile1148Thr,Gly1149Glu,3244-2A>C,3682A>T) three-dimensional structure model by three-dimensional structuretemplate of wild-type ATP7B protein. Three-dimensional structure of mutant proteincompared with wild-type ATP7B protein,the result showed that the three-dimensionalstructure ATP7B mutant protein has changed.The Arg778Leu, Ala874Ser,Ser921Arg,Ile1148Thr、Gly1149Glu mutant protein three-dimensional structure:the second and sixthtransmembrane helix structure extended by one residue，the length of two fold structure in thephosphorylationthe and a folded structure in the Transduction domain extended by oneresidue。These parts of the structural changes that may affect the normal copper transporter.3244-2A> C mutation lead to truncated abnormal ATP7B protein, three-dimensionalstructure lack of N domain (mostly),P domain,TM7domain and TM8domain;3682A>T mutation lead to truncated abnormal ATP7B protein too, three-dimensional structure lack of Pdomain (mostly),TM7domain and TM8domain; These two truncated proteins may be lostthe copper transport function completely.7． Splicing mutation3244-2A>C study by RT-PCR, sequencing results showed that:transcribed a lack of13base sequence of mRNA compare with normal secquence in mRNAlevel.The missing13bases sequence is located in the5’end of the15th exon.The basesequence of the missing number are non-multiple of3,will causing the change of the locationof the reading frame;Abnormal post-transcriptional mRNA sequence encountered terminatorat1115codons,translated a truncated protein. After collecting the clinical data and results ofgenetic testing of3244-2A> C,2333G> T heterozygous mutation and2333G> T homozygousmutation patients to study the genotype and clinical phenotype relationship betweenthem,the results indicated that3244-2A> C mutation is one of the prognosis of severemutations.8．By analyzing the splicing mutation3244-2A> C mutation location and base sequence ofexon15,On the3’end of the14th intron,the splice recognition sites AG Mutated to CG，Can’tbe Identification by splicing enzyme.In the5’end of the15th exon,the Twelfth andThirteenth base sequence is AG,become a A new splice site Identification by splicingenzyme.This is the ATP7B3244-2A> C splicing mutation molecular mechanism of theaberrant transcripts,that is One of the pathogenic mechanism of the Wilson disease.