Improvement And Application Of Gene Diagnosis Of DMD Patients And Carriers With Deletion Mutation

Objectives:1. To establish a rapid,accurate and visualized method for detecting DMD patient with deletion mutation.2. To optimize the molecular assays and establish a relatively perfect system for detecting DMD carriers,and provide more reliable genetic counseling for high risk population.Methods: 1. 35 probands of 35 DMD pedigrees were screened by multiple PCR,using 11 primers aiming at deletion mutation hotspots. PCR products were detected by agarose gel electrophoresis and denaturing high-performance liquid chromatography (DHPLC) analysis.2. DMD gene copy numbers of detected female relatives of the DMD pedigree with deletion mutation was relative quantitative assayed using competitive PCR and DHPLC.3. DMD gene copy numbers of detected female relatives of the DMD pedigree with deletion mutation was absolute quantitative assayed using real-time fluorescence quantitative PCR.4. The results of competitive PCR,DHPLC and real-time fluorescence quantitative PCR were analysised by SPSS13.0 statistical package. Results: 1. 18 probands of DMD pedigrees were detected having deletion mutation by multiple PCR followed by agarose gel eletrophoresis. The results of DHPLC analysis consistented with those of agarose gel eletrophoresis. Comparing to agarose gel eletrophoresis, DHPLC was much more sensitive, intuitive and accurate.2.DMD gene copy numbers of detected female relatives of the DMD pedigree with deletion mutation were decided by relative and absolute quantification assays using competitive PCR,DHPLC and real-time fluorescence quantitative PCR.13 carriers and 13 noncarriers were detected.7 DMD patients were de novo.Conclusions:1. For the combination of agarose gel electrophoresis and DHPLC,deletion mutation analysis was rapid and accurate,it can be applied in the diagnosis for DMD patients with deletion mutation.2. Combined relative and absolute quantification assays using competitive PCR , DHPLC and real-time fluorescence quantitative PCR, The molecular diagnosis system was improved.It is an accurate and high reliable method that is well suited for routine use in clinical laboratories.