Research On The Detection Of DYSTROPHIN By MLPA And The Regulation Of UTROPHIN Expression

IntroductionDuchenne and Becker muscular dystrophies(DMD and BMD) are X-link drecessive lethal disease with an incidence of～1 in 3500 newborns,and it has been estimated that approximately one third of the cases result from new mutations.As the disease progresses,the contractures increasingly develop,leading to the asymmetrical spinal deformities.Most patients die at the age of 20 of pneumonia related to chronic respiratory insufficiency.The allelic disorder BMD has a milder clinical course and a slower disease progression.With an incidence of～1 per 120,000 newborns.Patients with BMD have a reduced life expectancy,but the majority of patients survived as normal and the affected patients remain ambulant until 16 years of age,although onset in the 3^rd or 4^th decade,even later.Cardiac involvement is invariably associated with DMD and BMD,one third of DMD is associated with mild but potentially significant difficulties in a range of neurobehavioral areas.Besides,many patients have language handicap.IQ is below normal one percentage point.There is currently no effective treatment.so exploring the molecular mechanism of DMD/BMD will provide theories for improving genetic cohort,theatment of DMD/BMD.Multiplex Ligation-dependent Probe Amplification(MLPA) has become widely used for detecting deletions or duplications of the DYSTROPHIN gene among patients and carriers.It was established by Schouten based on MLPA.After DMD was characterized by the France sicientist Guillaume-Benjamin-Amand Duchenne in 1861,many genetist working for optimal management of DMD.Several curative therapeutic strategies including cell and gene therapy are being pursued but are still at an experimental stage.Although viral-mediated gene therapy has been at the forefront of the field,several non-viral gene therapy approaches have been applied to animal and cellular models of DMD. These include plasmid-mediated gene delivery,antisense-mediated exon skipping,and oligonucleotide-mediated gene editing.In the past several years,non-viral gene therapy has moved from the laboratory to the clinic.UTROPHIN might be able to serve as a surrogate for DYSTROPHIN in DMD muscle fibers because there is convincing evidence for functional redundancy between these two proteins.Indeed,UTROPHIN shares a high degree of sequence identity with DYSTROPHIN and also associates with members of the DAPC.Moreover,studies in the mdx mouse,a DYSTROPHIN negative model of DMD,have established that the elevation of UTROPHIN levels in dystrophic muscle fibers can restore sarcolemmal expression of DAPC members and alleviate the dystrophic pathology.This elevation can be accomplished by both germline gene transfer and somatic gene transfer of UTROPHIN.Together,these findings indicate that a gene-therapy approach focusing on induction of exogenously supplied UTROPHIN to DMD muscle fibers is a plausible treatment for this disorder.MethodsSamples from 59 families were collected with informed consent.All subjects were referred from the Department of Pediatrics,Shengjing Hospital of China Medical University and the Department of Medical Genetics,Peking Union Medical University between January 2005 and December 2007.The research plan was approved by the ethics committees of both universities.The subjects included 51 boys diagnosed with DMD and 8 with BMD,their unaffected parents and 19 amniotic samples.Clinical diagnosis was made based on physical examination,family history,serum creatine phosphokinase(CPK) levels,age of onset,calf pseudohypertrophy,wheelchair confinement,presence of cardiomyopathy and electromyographic patterns.Multiplex PCRThe DMD gene of 59 patients were detected by multiplex PCR.MLPA analysis The mutations of DMD gene were detected by MLPAStatistical analysisEfficacy of deletion/duplication detection by MLPA as compared with that of mPCRPrenatal diagnosisFor the 19 couples who were at risk of carrying another DMD/BMD fetus, prenatal diagnoses were achieved for all subjects using combined MLPA and linkage analysis.P-Match software was used to analyze the sequence upstream of the transcription start site of the UTROPHIN gene.The 1500bp sequence uptream of UTROPHIN gene was obtained from http://www.ensembl.org,we used P-Match software to predict the binding sites of transcriptional factors on the sequence.Chromatin immunoprecipitation assay and Electrophoretic mobility shift assayThe chromatin was extracted from Hela cells,sheared with an Enzymatic Shearing Kit to obtain 500-1000bp fragments.EN1 antibody was used at the immunoprecipitation step.Eluted DNA from the sample and control was assessed for the presence of UTROPHIN DNA region by PCR.Nucleoprotein was extracted from Hela cells and incubated with the UTROPHIN upstream region-containing binding site 2 labeled using a Biotin 3′End DNA ing Kit for 60 min at room temperature in a gel shift buffer.Reactions were examined for nucleoprotein binding by electrophoretic mobility shift assays(EMSA) on a 10% nondenaturing polyacrylamide,transfered to the memberane,cross-linked.DNA binding bands were detected using a chemiluminescence system.siRNA EN1 was knockout by siRNA,the expression of UTROPHIN were detected by Real time PCR.ResultsMultiplex PCR31 exon deletions were detected by multiplex PCRMLPA analysis33 exon deletions,6 exon duplications and one point mutation were detected by MLPA.Statistical analysis10 mutations including 3 exonic deletions,1 single base deletion and 6 exonic duplications were only detected by MLPA.16 deletions in fact had involved more exons.Prenatal diagnosis5 were at risk of DMD/BMD,4 carriers,10 normal fetus were detected by MLPA and STR analysis in 19 couples who were at risk of carrying another DMD/BMD fetus.P-Match analyze resultsThe 5' region of the human UTROPHIN gene contained two potential binding sites for the EN1 protein designated EN1 binding site 1(-1074～-1080) and EN1 binding site 2(-892～-899).EN1 binds with site 2 in UTROPHIN promoter region in the developing limb in Hela cells.EN1 directly binds to site 2 in UTROPHIN promoter region.To verify the binding in vivo of EN1 to binding site 2 within the UTROPHIN promoter,we used the chromatin formaldehyde cross-linking and immunoprecipitation (CHIP) technique.The immunoprecipitated Hela cell chromatin showed a substantial enrichment only of the sequence containing site 2,indicating that EN1 efficiently bound only to site 2 in vivo.No enrichment was detected for the control site.SiRNAAfter EN1 was knockout by siRNA,the expression of UTROPHIN was increased.Conclusion1.For the comprehensive coverage of all exons of the DYSTROPHIN gene,compared with that of mPCR MLPA should be the method of choice for initial screening of DMD/BMD patients.2.For its sensitivity and robust performance,MLPA can provide a powerful assay for prenatal diagnosis for such diseases.3.EN1 may decreased the expression of UTROPHIN...