Study On The Hotspot Mutations Of The Wilson Disease Gene In Hunan And Multiplex PCR Reverse Hybridization Technology

Objective:Establishing a method for directly sequencing all the exons of ATP7B gene.Through using this method,investigating the style and frequency of mutations and polymorphisms of ATP7B gene,analyzing the characteristics and hot point mutations of ATP7B gene exons and exon-intron junction in Hunan Han patients with wilson's disease(WD).Establishing a method which combines multiplex polymerase chain reaction(PCR)and reverse hybridization for rapid examining the ATP7B gene mutations.And assaying this method's feasibility,sensitivity and specify.Methods:1.Screening the hotspot mutations of the Wilson disease gene(ATP7B).The genomic DNAs of 32 WD patients were extracted from peripheral blood leukocytes,and the ATP7B gene(exon1～exon21)were amplified by PCR.Then,the PCR products were directly sequenced and analyzed by BLAST.2.Screening the mutations of ATP7B gene by using multiplex PCR and Reverse hybridization technology.Firstly,four probes for wild-type G2333(R778)and G2855(A952)as well as mutants T2333(L778)and A2855(K952)were designed,then,the probes with several control probes were coated and immobilized in a membrane.Secondly,multiplex PCR containing biotin-labeled primers was used to amplify ATP7B exons 8 and 12 from DNA samples.Thirdly,denatured biotin-labeled amplicons containing ATP7B exons 8 and 12 were hybridized with membranes coated with the specific probes,then,by using streptavidin-conjugated alkaline phosphatase,the hybridation signals were visualized by colorimetric detection with nitroblue tetrazolium and 5-Bromo-4-chloro-3-indolyl phosphate(NBT/BCIP).3.Comparing the feasibility of the combination of multiplex PCR and Reverse hybridization technology with direct sequencing.Results:1.Established a method to amplify the entire ATP7B gene's exons and exon-intron borders sequences.PCR products were confirmed by directly sequencing and blasting with the Genbank reference genome sequences. The result suggested that the primer designing and experimental conditions are appropriate and reliable.2.29 of 32 cases(90.63%)of WD patients showed mutations or polymorphisms in ATP7B gene exons.Among 32 patients,21 showed pathogenic gene mutations(65.63%),7 patients showed compound heterozygous mutations(53.13%).Only 4 patients showed homozygous mutation(polymorphism Arg952Lys),accounting for 12.50%.3 patients had not showed any mutations or polymorphisms.3.Six different pathogenic gene mutations had been found in this studying:6.25%of patients showed heterozygous mutation 994G→T (Glu332Ter)in exon2;40.63%showed heterozygous mutation 2333G→T (Arg778Leu)in exon8;3.13%showed heterozygous mutation 2828G→A (Gly943Asp)in exon12;3.13%showed heterozygous mutation 2975C→T(Pro992Leu)in exon13;6.25%showed heterozygous mutation 3532A→G(Thr1178Ala)in exon16;9.38%showed heterozygous mutation 3884C→T(Ala1295Val)in exon18.Among them,994G→T,3532A→G,3884C→T were novel mutations firstly reported by us.Also in this studying,six polymorphisms had been found:3.13%of patients were found with 1122C→G(Val1374Val)polymorphism in exon2;6.25%were 1216T→G (Ser406Ala)polymorphism in exon2;6.25%were 1366G→C(Val456Leu) polymorphism in exon3;40.63%were 2250C→G(Leu770Leu) polymorphism in exon 8;31.25%were 2855G→A(Arg952Lys) polymorphism(18.25%heterozygous and 12.50%homozygous)in exon12; 6.25%were 3419T→C(Val1140Ala)polymorphism in exon16.Among of them 1122C→G polymorphism in exon2 were firstly reported by us.No mutations were found in Exon-1,exon-4,exon-5,exon-6,exon-7, exon-9,exon-10,exon-11,exon-15,exon-17,exon-19,exon-20 and exon-21.4.Although all exons of ATP7B gene had been sequenced,there are still 3 patients did not show any mutations,and 9 patients showed only polymorphism without any pathogenic mutations.Since many patients had not showed mutations or only one heterozygous mutation in exons,it strongly suggests that the mutations of ATP7B gene which causes Wilson's Disease may be located in other regions besides exons.5.Established a screening method for ATP7B gene exon8 Arg778Leu and exon12 Arg952Lys mutations by combining multiplex PCR and reverse hybridization technology.Probes for G2333(Arg778),G2855(Arg952),T2333(Leu 778)and A2855(Lys 952)were coated and immobilized in a membrane,then were hybridized with the multiplex PCR products which contained biotin-labeled ATP7B exons 8 and 12 from patients' gDNA. Control experiments did not result in false positive and false negative.6.By using the method mentioned above,32 WD patients were screened.13 patients showed heterozygous mutation Arg778Leu(2333G→T)in exon 8,6 patients showed heterozygous polymorphism 2855G→A (Arg952Lys),4 patients showed homozygous mutation 2855G→A.The results are same to the direct sequencing results.No false positive and negative were found.Comparing to direct sequencing,reverse hybridization was highly sensitive and specific.Conclusion:1.We established a method for amplifying and sequencing all of the 21 exons and exon-intron junction of ATP7B gene.2.29 of 32 cases(90.63%)of WD patients showed mutations or polymorphisms in ATP7B gene exons.21 of 32 patients were found with pathogenic gene mutations(65.63%).Among the 29 mutaions, heterozygous mutations account for 53.13%and homozygous mutations account for 12.50%.3 patients had not showed any mutations and polymorphisms.3.Six different pathogenic gene mutations had been found in this studying:994G→T(Glu332Ter),2333G→T(Arg778Leu),2828G→A(Gly943Asp),2975C→T(Pro992Leu),3532A→G(Thr1178Ala)and 3884C→T(Ala1295Val).Among them 994G→T,3532A→G,3884C→T were novel mutations firstly reported by us.Also in this studying,six polymorphisms had been found.Among of them,1122C→G polymorphism in exon2 were firstly reported by us.Most mutations of ATP7B exons are heterozygous and only 4 patients showed homozygous mutaions(12.50%).4.The Hunan Han patients with Wilson's Disease showed a wide spectrum.Heterozygous spot mutations contribute most of the cases. ATP7B gene mutations vary in different regions.Arg778Leu is the hottest point mutation of ATP7B gene(40.63%)in Hunan area Han patients,and Ala1295Val(9.38%)is the second in this studying.5.Since many patients had not showed mutations or only one heterozygous mutation in exons,it strongly suggests that the mutations of ATP7B gene which causes Wilson's Disease may be located in other regions besides exons.6.We established a screening method for ATP7B gene exon8 Arg778Leu and exon12 Arg952Lys mutations by combining multiplex PCR and reverse hybridization technology.7.Comparing to direct sequencing,reverse hybridization was highly sensitive and specific.Both its specificity and sensitivity were 100%. Multiplex PCR and reverse hybridization technology are simple,fast, sensitive and specific.On the basis of known hot mutations of ATP7B gene in a certain area,we conclude that it is a useful and reliable tool for WD diagnosis and screen.