Gene Mutation Analysis Of Duchenne Muscular Dystrophy And Tuberous Sclerosis Complex

IntroductionDuchenne muscular dystrophy(DMD) is a devastating X-linked recessive dystrophinopathy,characterized by rapidly progressive degeneration and necrosis of proximal muscles and calf pseudo-hypertrophy.DMD is caused by the changes in the structure and function of dystrophin protein with a world-wide incidence of 1 in 3500 live male birth.Females are usually carriers and no symptoms.To date,no effective treatment is available.The dystrophin gene is the only causative gene for DMD,which is located on Xp21.2,consists of 79 exons and encodes the dystrophin protein.The large size of the dystrophin gene is liable to a high incidence of de-novo mutations and ectopic breakpoints.In specific,approximately 55-65%of DMD cases are associated with one or more exons deletions clustered in 2 hotspot regions in 5'terminus and around exon 44-53 of the gene.And about 5%-10%of DMD mutations are associated with duplications,whereas the remaining 25-30%cases may be caused by point mutations and small insertions/deletions sporadically.Due to the large size and complex mutation types of the dystrophin gene,it is a challenge to establish an effective and full-scale molecular diagnosis method.A number of techniques for detecting mutations in DMD patients have been developed such as multiplex PCR(mPCR),Southern blot,real-time quantitive PCR, Fluroscence in situ hybridization(FISH) and mltiplex amplifiable probe hybridization(MAPH).However,the above methods are all have some disadvantages in screening dystrophin mutations.The most common is mPCR which cannot detect duplications and carriers status,further more it cannot cover the whole 79 exons. Recently,a new technique,multiplex ligation-dependent probe amplification(MLPA), has been depicted by Schouten as a first screening,which allows the detection of large genomic rearrangements(deletions/duplications) in the dystrophin gene by simultaneous amplification of up to 40 target sequences.Additionally,denaturing high-performance liquid chromatography(DHPLC) has been widely used in gene diagnosis of genetic disorders.In this study,we chose MLPA and DHPLC,assisted with STR linkage analysis to detect DMD patients,carriers and high risk fetuses.For the fetus in which no deletion/duplication was found by MLPA and with an apparent family history,we used STR linkage analysis to judge whether it is affected or a carrier. After that,DHPLC was employed to find specific point mutations.Our study was aimed to develop a comprehensive and rapid strategy,which will be valuable in improving the availability of molecular testing for DMD patients.Materials and Methods1.Materials46 unrelated suspectable male DMD patients at the age of 1.5month-16 years old, 6 female carriers,from the Pediatric Department,the 2^nd Affiliated Hospital of China Medical University,entered the study.3 high risk fetuses from the Research Center for Genetics of China Medical University,with peripheral blood,cord blood or amniotic fluid samples of 3 core families.Genomic DNA of 16 patients negative for MLPA,11 Male and 5 females,were supplied by Institute of Basic Medical Science Chinese Academy.of Medical Science.10 unaffected males and 10 unaffected females without a DMD family history were recruited in the study as the controls.Genomic DNA was extracted from peripheral blood samples of the patients and controls using a standard proteinase K digestion and phenol-choloroform extraction procedure.Some blood samples,one cord blood and 2 amniotic fluid were extracted using Universal Genomic DNA Extraction Kit Ver.3.0(TAKARA,Japan).MLPA DMD KIT(SALSA MLPA KIT P034/035 DMD/BMD) was purchased from the MRC-Holland(Amsterdam, Nertherlands).The main facilities included UNOⅡ48 PCR thermal cycler(Biometra, German),Beckman CEQ-8000 genetic analytic system(Beckman,USA) and WAVE^? nucleotide fragment analysis system(Transgenomic,USA).2.Methods(1) Detection of deletions/duplications in the dystrophin gene by MLPAMLPA was performed using the MLPA DMD KIT.MLPA probes are divided into two probemix,P034 and P035,including probes for each exon sequence.Briefly, 50-500 ng of genomic DNA in a volume of 5μL Tris-EDTA was denatured(98℃,5 min),cooled and mixed with MLPA P034 or P035 probemix.The mixture was heated to 95℃for 5 min and then incubated at 60℃overnight for probe hybridization.After 16h,ligation was performed with Ligase-65 enzyme at 54℃for 15 min and Ligase-65 enzyme was inactivated at 98℃for 5 min.Then PCR was performed with the specific SALSA FAM PCR primers.Amplification products and Size Standard 600 were mixed completely as 80:1.The mixture were run and analyzed by capillary electrophoresis on the Beckman CEQ-8000 genetic analytic system.The peaks obtained after capillary electrophoresis were analyzed by Fragment Analysis software and processed by Coffalyser version 9.0(http://www.mlpa.com/coffalyser/download.html),so as to figure out the relative peak ratio(RPR) for every single exon and its corresponding bar chart. 10 unaffected males and 10 unaffected females were included in the analysis as controls.Any result with obvious single-exon deletion was validated by further PCR and sequencing and any single exon duplication was confirmed by 3 repeated experiments.(2) Detection of small mutations in the dystrophin gene by DHPLC and sequencing16 Patients negative for MLPA analysis in the dystrophin gene were subjected to further DHPLC screening.Exons were separately amplified with primers designed to cover all of the 79 exons and their flanking sequences.Unpurified PCR amplicons from patients were mixed with those from male controls(1:1) and then denatured,followed by cooling down in a thermal cycler.DHPLC was performed on the WAVE^? system.The pre-treated amplicons were processed at the optimal separation gradient and temperature determined by the WAVEMARKER 4.1 software.If a PCR amplicon presents a chromatogram different from the control(wild type) in shape or retention time,the corresponding exon was directly sequenced to identify the position and the type of the mutations using the Beckman CEQ8000 DNA genetic analytic system.We check the DMD database (www.dmd.nl) to find whether the variation is a known mutation.The National Center for Biotechnology database of genetic variation(dbSNP) was queried to identify the existence of the common SNPs.(3) Prenatal diagnosis with combined MLPA and STR linkage analysisAmniocentesis was performed at gestation 16-22 weeks under monitoring of type-B ultrasonic.Centrifuge 15ml amnionic fluid 4000r/min for 8 min.Then discard the supernatant and wash the precipitation 3 times with physiologic saline.The precipitation was collected and added another 200μL physiologic saline.After that genomic DNA was extracted with Universal Genomic DNA Extraction Kit Ver.3.0 bought from TaKaRa company(Japan).We Performed MLPA analysis for proband, carriers and fetus,the method was the same as described above.STR analysis was carried out for families in which all the members including proband,carrier and fetus were all negative for MLPA.We selected 5 STR markers scanning the whole dystrophin gene and its flanking sequence.Primers were designed and PCR was performed.,followed by a denaturing polyacrylamide gel electrophoresis. Subsequently we analyzed the allele type according to the above electrophoresis result. Results1.MLPA results of DMD patients and carriersCopy number variation of one or more exons of the corresponding MLPA products were identified in 24(52.2%)out of the 46 cases.15 cases showed multiple exon deletions,5 cases single-exon deletion,and 4 duplications was identified in all patients. All cases with single-exon deletion were confirmed in the uniplex PCR except the deletion of exon 58 in patient D3 and his mother,on which the sequence analysis revealed a point mutation c.8608C＞T(p.Arg2870X) that was predicted to produce a premature stop codon.5 out of 6 suspect carriers were identified carrying the same mutation as her affected relatives by MLPA.2.Results of DHPLC and sequencing79 exons covering two deletion hotspots and the 3'UTR region were amplified.A total of 16 cases,including 11 male patients,5 female patients.All 16 cases occurred abnormal peak shapes.But no causative mutations were identified by sequencing,Only to find some genetics variation.3.Prenatal diagnosis results of DMD high risk fetusesWe used MLPA analysis to test the 3 high risk fetuses and their family members. The three fetuses are all female according to our results,and we found a deletion of exon 44 in the proband of Family 1.Then the result was confirmed by a uniplex PCR. STR linkage analysis of the 2 families shows the 2 female fetuses were not potential carriers and had low risk to be affected.Conclusion1.We found 20 deletions and 4 duplications in 46 DMD patients,3 deletions and 1 duplication in 6 carriers which was carrying consistent with their affected relatives. Therefore,MLPA could detect deletions,duplications and female carriers of dystrophin gene easily,covering all the exons and it can also refine the breakpoint to a specific intron.MLPA was simple to manipulate and the result is sensitive and reliable.In conclusion,MLPA was proven to be a powerful tool for the detection of dystrophin gene.2.A combination of MLPA,STR linkage analysis and DHPLC could provide a comprehensive and full-scale strategy for screening DMD causative mutation and DMD prenatal diagnosis,which has a widely clinical application.