连接片段在缺失型假肥大性肌营养不良症产前诊断应用的初步研究OA

Objective To preliminarily explore the application value of junction fragments in the prenatal diagnosis of deletion-type dystrophinopathies[including Duchenne muscular dystrophy(DMD)/Becker muscular dystrophy(BMD)].Methods A female carrier from a BMD pedigree who has been treated multiple times at the Southern Hospital of Southern Medical University from 2005 was selected as the research subject.The proband of this pedigree was confirmed to have a deletion of exons 3-5 in the DMD gene,and the deletion junction fragment had been cloned.A pair of new primers,D4-F/R,was designed as close as possible to the breakpoints to analyze and detect the junction fragment sequence of this pedigree.Chorionic villus sample A(from the first induced abortion)and chorionic villus sample B(from the second induced abortion)were collected from the subject following two episodes of unintended pregnancy that resulted in induced abortion,and genomic DNA was extracted and prepared from these samples.Polymerase chain reaction(PCR)was performed using primers D4-F/R to detect the junction fragment in the genomic DNA of the two chorionic villus samples.PCR detection for gene deletion was conducted using exon 3 primers ex3-F/R,while gender identification was carried out using primers SRY-109F and SRY-245R.Results For the genomic DNA of Chorionic villus sample A(from the first induced abortion),the detection of the junction fragment showed amplification of a positive fragment consistent with that of the proband.The detection of exon 3 resulted in positive amplification without deletion.Gender identification indicated a female.Thus,chorionic villi A was diagnosed as a female carrier of BMD.For the genomic DNA of Chorionic villus sample B(from the second induced abortion),the detection of the junction fragment showed negative amplification.The detection of exon 3 showed positive amplification without deletion.Gender identification indicated a male.Therefore,chorionic villi B was diagnosed as a normal male fetus.Conclusions In this study,chorionic villus samples obtained from two induced abortions of the study participant were used to simulate the prenatal diagnostic process for deletion-type DMD/BMD.Conventional PCR was applied to simultaneously detect disease-related gene junction fragments and exons in the corresponding deletion regions in fetal specimens from female DMD/BMD carriers,combined with sex identification.The feasibility of further clinical application of this method was also proposed.