Application Of Novel HRM Technology In Genetic Disease And Pharmacogenomics

Background: High resolution melting(HRM)is a kind of nucleic acid analysis technology based on melting curves.Because of its easy to operate,fast and low cost,HRM technology has been widely concerned since its development.New detection methods based on HRM are also emerging.Williams-Beuren syndrome is a kind of serious and common multisystem disorders caused by deletion of contiguous genes at 7q11.23,whereas duplication of the same area may cause multisystem disorders as well.Clopidogrel is widely used in antiplatelet therapy in patients undergoing percutaneous coronary intervention to reduce ischemic complications,but the polymorphism of CYP2C19*2,CYP2C19*3,CYP2C19*17,PON1 Q192 R and ABCB1 C3435 T may contribute to clopidogrel resistance.Quetiapine and aripiprazole are two kinds of sencond-generation antipsychotics,the treatment effect and side effect of which were affect by CYP1A2*1F,CYP3A5*3 or CYP3A4*1G,ANKK1 Taq1 A respectively.Objective: dNTP-limited PCR with HRM was adopted to develop new methods to detect 7q11.23 microdeletion/microduplication.5' GC rich tails on primers and multiplex PCR was used to develop new methods to genotype the 5 clopidogrel resistance associated SNPs.Multicolor probe HRM was adopted to develop new methods to genotype the 4 SNPs related to quetiapine and aripiprazole metabolize.Results:(1)A new genotyping method for detecting 7q11.23 microdeletion/microduplication was developed based upon dNTP-limited PCR with HRM.Samples from 3 WBS region duplication syndrome patients,15 WBS patients and 18 healthy individuals were genotyped by this method in a blinded fashion,and the sensitivity and specificity was 100%,which was proved by CytoScan HD array.(2)A new genotyping method for detecting 5 clopidogrel resistance associated SNPs was developed based upon multiplex HRM.912 samples from consecutive acute coronary syndrome patients were used for a blinded study of multiplex HRM.The sensitivity of HRM was 100% and the specificity was 99.93%,which was proved by Sanger Sequencing.(3)A new genotyping method for detecting 4 SNPs related to quetiapine and aripiprazole metabolize was developed based upon multicolor probe HRM.240 samples from healthy people were used for a blinded study of multicolor probe HRM,and the sensitivity and specificity was 100%,which was proved by Sanger sequencing.Conclusion: The three methods we developed based upon HRM were fast,accuracy,low-cost and easy to operate,and has a good practical application value.