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1、论著 Prenatal molecular diagnosis of Duchenne and Becker muscular dystrophy Qing LI 1 ,Shao-ying LI1,Dong-gui HU1,Xiao-fang SUN1,Dun-jin CHEN1,Cheng ZHANG2,Wei-ying JIANG2 (1. Guangzhou Second People s Hospital,Guangzhou research institute of Obstetrics and Gynecology,Guangzhou 510150, China; 2. Basic
2、 Medical College,Sun Yat-Sen University) ABSTRACT Objective:Duchenne and Becker muscular dystrophy(DMD/ BMD)is an X-linked lethal recessive disease caused by mutations in the dystrophy gene. There is no efficient treatment for this seri- ous and disabling disease. We established a combination method
3、 to detect carriers and perform prenatal diagnosis. Methods:In our study,from 1994 to 2005,using a different combination of 5 methods,in- cluding SRY gene amplification,multiplex PCR,multiplex Fluorescence PCR capillary electrophoresis, multiplex ligation-dependent probe amplification(MLPA)and linka
4、ge analysis of short tandem repeats (STR) ,36 prenatal diagnosis were performed for pregnancies at risk of having a DMD/ BMD baby through amniocentesis. Results:Fourteen out of 21 male fetuses were found to be affected and respective pregnancies were terminated. A combined diagnostic rate of 83% was
5、 achieved for 30 cases with dele- tions,duplications,and non-deletion mutations after tested by more than one method. Conclusion:U- sing a combined method,we can diagnoses patients and carriers in DMD families,and perform prenatal diagnosis for the risk fetus. MLPA provides a simple,rapid and accura
6、te method for deletions and dupli- cations of all the 79 DMD exons. MLPA method for DMD diagnosis is the first report in our country. KEY WORDS Muscular dystrophy,Duchenne;Prenatal diagnosis;Gene;Nucleic acid amplification techniques DuchenneandBeckermusculardystrophy (DMD/ BMD)is an X-linked lethal
7、 recessive genetic disease caused by mutations in the dystrophin gene lo- catedontheshortarmoftheXchromosome (Xp21) 1. It is one of the most serious and common inherited neuromuscular diseases,affecting approxi- mately 1 in 3 500 newborn males. In a typical affected male,clinical symptoms arise arou
8、nd the age of 3 with progressive muscle weakness. The patient is non ambu- latory by the age of 9 or 10 and usually dies by 20, fol- lowing cardiac or respiratory complications. BMD is a milder form characterized by a later and slower disease course,with death generally occurring in the age of 30 or
9、 40. The mothers are generally asymptomatic carriers who pass on the disease. Presently,there is no efficient treatment for this serious and disabling disease. Genetic counseling and molecular diagnosis that allow the identification of car- riers are therefore the only solutions offered to DMD famil
10、ies. Carrier detection and DNA-based prenatal di- agnosis may be performed when the disease-causing mutation has been detected in an affected individual. In this study,we report our molecular diagnosis results from 36 families with DMD risk fetus,all data were collected from 1994 to 2005 in our hosp
11、ital. 1 Patients and Methods 1. 1 Patients source From 1994 to 2005,prenatal diagnosis was per- formed in our hospital for 36 pregnant women at risk of having a DMD/ BMD baby. Most patients were referred to us from our own genetic counseling clinic and the re- mainders from genetic counseling clinic
12、 of either De- partment of Medical genetics or Department of Neurolo- gy of SunYat-sen University. Probands in all families exhibited canonical clinical manifestations of DMD or BMD and had classical pathological changes in muscu- lar biopsy,the activity of serum muscular creative ki- nases and Elec
13、tromyography(EMG) ,the transmission pattern is X-linked trait,excluding other similar neuro- muscular system disorders. 1. 2 DNA extraction method For prenatal diagnosis,10 mL of amniotic fluid was obtained from each pregnant woman at gestation of 16 - 22 weeks via amniocentesis and centrifuged at 4
14、 000 r/ min to collect cell pellets. For linkage analy- sis,1 mL of peripheral blood sample was collected from every proband and some relevant relative for DNA extraction. Before 2 000,Cell pellets were lysed and digested in solution containing proteinase K,then ex- tracted using a phenol/ chlorofor
15、m method,and finally dissolved in TE buffer;After 2 000,DNA extraction was carried out using QIamp DNA mini kits( Qiagen, German) . 1. 3 Gender determination for amnionic fluid Genomic DNA samples of normal male(46, XY) and female(46, XX)were used as controls. Amplify DMD exon 6 as internal standard
16、,test the amnionic fluid sample s SRY gene. Each sample amplifies two tubes:(1)SRY tube and(2)SRY + DMD exon 6 internal reference (Figure 1) . 1. 4 Multiplex PCR Multiplex DNA amplifications of the dystrophin gene were carried out according to Chamberlain and Beggs methods,using two multiplex PCR as
17、says allo- wing the amplification of 9 exons each:exon 4, 8, 12, Corresponding author s e-mail, qli2006 yahoo. com 35 北京大学学报 (医学版) JOURNAL OF PEKING UNIVERSITY (HEALTH SCIENCES) Vol.38 No. 1 Feb. 2006 17, 19, 44, 45, 48, 51 and exon 3, 6, 13, 43, 47, 50, 52, 60, 49. The PCR products were run on 2% a
18、garose gel and the bands visualized by staining with ethidium bro- mide,photographed for records 1 -3. 1. 5 Multiplex fluorescence PCR capillary electropho- resis analysis In this method,two fluorescently-labeled multi- plex PCR assays were developed. These two assays am- plify 18 exons from the del
19、etion / duplication hotspot of the dystrophin gene. The combination of fluorescent multiplex PCR and the Gene Scan software of the ABI 310 Genetic analyzer allow each of these 18 PCR prod- ucts to be accurately sized and quantitated according to the peak location and area(Figure 2) 2 -4. Electrophor
20、ese on 2% agarose gel; 510 bp, SRY amplify fragment;200 bp, DMD exon 6 internal reference; M, marker; 1, amniotic fluid sample amplify SRY gene and DMD exon 6;2, amniotic fluid sample amplify SRY gene; 3, male control DNA amplify SRY gene and DMD exon 6;4, male control DNA amplify SRY gene. Figure 1
21、 Analysis of SRY gene amplification (family34:amniotic fluid sample is female) 1. 6 Multiplex ligation-dependent probe amplification (MLPA) The MLPA reactions for probe sets SALSA P034 and P035(MRC-Holland Amsterdam)were performed essentially as described. Ligation was performed with the Ligase-65 e
22、nzyme, the ligated products were ampli- fied by PCR according to the manufacturer s protocol using one primer labeled with 6-FAM. The resulting fragments were analyzed on the ABI 310/3100 capillar- y genetic analyzer using the Genescan software. The si- zes of exon-specific peaks were identified acc
23、ording to their migration. The power of the MLPA-analysis is that it screens all 79 exons of the DMD gene for dele- tion and duplication in two reactions(Figure 3) 5, 6. Electrophoresis analysis(Family 23:DMD exon45 duplication) ; A, fa- ther;B, mother(carrier) ;C, fetus(affected) ;D, normal female;
24、E, normal male. Figure 2 Analysis by multiplex fluorescence PCR capillary A, normal reference;B, proband;C, carrier;D, normal fetus. Figure 3 Analysis by MLPA( Family 19:DMD exon 45-50 detetion) 1. 7 Linkage analysis with PCR-amplified short tan-dem repeats(STRs) 45 北京大学学报 (医学版) JOURNAL OF PEKING UN
25、IVERSITY (HEALTH SCIENCES) Vol. 38 No. 1 Feb. 2006 Select 4 STR markers to analyze DMD non-dele- tion form family. If mutating loci can not be found, but family history has DMD records,the STR family link- age study and analysis will be a helpful assay. 2 Results In this study,36 prenatal diagnosis
26、were per- formed for women at risk of having a DMD/ BMD baby at gestation of 16 - 22 weeks through amniocentesis. This was achieved by using a different combination of 5 different methods,including SRY gene amplification, multiplex PCR,multiplex Fluorescence PCR capillary electrophoresis, MLPA and l
27、inkage analysis of STR. Mutations of DMD gene found in probands of the 36 DMD/ BMD families analyzed in this study were shown in Table 1. 33%(12/36)of mutations were non-deletion mutations,61%(22/36)were deletions and the remainder 6%(2/36)was duplications. 36 prenatal diagnosis were carried out and
28、 results were given in Table 2. Sexing by SRY was performed for all 36 cases,21 were found to be male and 15 were fe- male. Out of the 36 amniotic fluid samples tested in this study,13 were analyzed by multiplex PCR,4 by multiple fluorescent PCR combined with capillary elec- trophoresis,8 by multipl
29、ex ligation-dependent probe amplification,and 12 by linkage analysis of short tan- dem repeats(STR) . Fourteen out of 21 male amniotic fluid samples were found to be affected and hence the respective pregnancies were terminated,the rest 7 nor- mal male pregnancies were continued. Out of 15 preg- nan
30、cies bearing a female fetus,only 2 were terminated upon requests. Out of 36 cases in this study,a com- bined diagnostic rate of 83% was achieved for 30 cases with deletions,duplications,and non-deletion muta- tions after tested by more than one method;a simple sexing was employed in 6 cases where al
31、l 4 STR loci were not informative. To our knowledge,this is the first DMD/ BMD prenatal diagnosis application report in China by use of the method of multiplex fluorescence PCR combined with capillary electrophoresis analysis and the method of MLPA. Table 1 Mutations of DMD gene found in probands of
32、 the 36 DMD/ BMD families Gene mutation formCasePercentage(%) Exon 8 deletion38. 33 Exon 17 deletion38. 33 Exon 45 deletion38. 33 Exon 48 deletion513. 89 Exon 19 deletion25. 56 Exon 44 deletion25. 56 Exon 45-50 deletion12. 78 Exon 47-52 deletion12. 78 Exon 48-50 deletion12. 78 Exon 49-52 deletion12.
33、 78 Exon 45 duplication12. 78 Exon 49-55 duplication12. 78 Nondeletion1233. 33 Total36100 Table 2 Testing results and clinical results of the 36 prenatal diagnosis No.ProbandFetus Assay SRYmPCRfmPCRMLPASTR DiagnosisClinical result 1Exon 8 delExon 8 +MaleLow riskBirth 2Exon 8 delExon 8 delMaleAffecte
34、dInduced abortion 3Exon 8 delFemaleNormalLow riskBirth 4Exon 17 delFemaleNormalLow riskBirth 5Exon 17 delExon 17 delMaleAffectedInduced abortion 6Exon 17 delFemaleNormalLow riskBirth 7Exon 45 delExon 45 delMaleAffectedInduced abortion 8Exon 45 delExon 45 delMaleAffectedInduced abortion 9Exon 45 delF
35、emaleCarrierCarrierInduced abortion 10Exon 48 delExon 48 delMaleAffectedInduced abortion 11Exon 48 delFemaleNormalLow riskBirth 12Exon 48 delFemaleNormalLow riskBirth 13Exon 48 delExon 48 +MaleLow riskBirth 14Exon 48 delExon 48 delMaleAffectedInduced abortion 15Exon 19 delExon 19 delMaleAffectedIndu
36、ced abortion 16Exon 19 delExon 19 +MaleLow riskBirth 17Exon 44 delExon 44 delMaleAffectedInduced abortion 18Exon 44 delExon 44 +MaleLow riskBirth 19Exon 45-50 delExon 45-50 +FemaleLow riskBirth 20Exon 47-52 delExon 47-52del HetFemaleCarrierBirth 21Exon 48-50 delExon 48-50del HetFemaleCarrierBirth 22
37、Exon 49-52 delExon 49-52 +MaleLow riskBirth 23Exon 45 dupExon 45 dupMaleAffectedInduced abortion 24Exon 49-55dupExon 49-55 dupMaleAffectedInduced abortion 25NondeletionFemaleCarrierCarrierInduced abortion 26NondeletionMaleNormalLow riskBirth 27NondeletionFemaleNormalLow riskBirth 28NondeletionMaleCa
38、rrierAffectedInduced abortion 29NondeletionMaleNormalLow riskBirth 30NondeletionMaleCarrierAffectedInduced abortion 31NondeletionMaleAffectedInduced abortion 32NondeletionMaleAffectedInduced abortion 33NondeletionFemaleFemaleBirth 34NondeletionFemaleFemaleBirth 35NondeletionFemaleFemaleBirth 36Nonde
39、letionFemaleFemaleBirth 55黎 青, 等 假肥大型肌营养不良症的产前基因诊断 3 Discussion DMD/ BMD is one of the most serious and com- mon inherited neuromuscular diseases.The mothers are generally asymptomatic carriers who pass on the disease. The progress of genetic technology and the ap- plication of new approaches for DN
40、A analysis may cur- rently be the only approach for prevention,through ge- netic counseling and prenatal diagnosis in the absence of therapy. Previously, the only intervention possible for fami- lies was genetic counseling for women at risk based on serum creatine kinase(CK)determinations. However,
41、this strategy was of limited value because it did not permit a definite conclusion as to carrier status. At the time,many couples abstained from having children or used the only type of prenatal diagnosis possible in this situation-fetal gender determination,which sometimes led to the aborting of ma
42、le fetuses,even through the probability of their being affected was at most 50% and could not be determined with certainty. Advances in molecular genetics led to the identifi- cation of the gene and its product,dystrophin. DMD gene is the largest known gene,spanning approximate- ly 2. 4 Mb of genomi
43、c sequence on Xp21,consisting of at least 79 exons. Its transcript is a 14 kb mRNA enco- ding the dystrophin protein. The dystrophin protein is absent or drastically reduced in the muscles of DMD patients,while it is present but reduced in BMD pa- tients. Large rearrangements in the gene are found i
44、n about two thirds of DMD patients,with approximately 60% carrying deletions,5% - 10% carrying duplica- tions and the remaining caused by point mutations and small inserting / deletions 7 -9. Several approaches for detection have been pub- lished,where the multiplex PCR developed by Beggs and Chambe
45、rlain,are the most commonly used for a clinical diagnostic set up. These methods allow a de- tection of 90% - 95% of the deletions. Quantitative PCR analysis allows for the PCR products to be accu- rately sized and quantified by capillary electrophoresis. These methods also allow detection of duplic
46、ations, and identification of carriers of deletions and duplica- tions. Recently,the MLPA method was introduced, these two set of probes allow a scanning for deletions and duplications of all the 79 DMD exons in just two PCR reactions. Furthermore,we have been able to i- dentify carries within the f
47、amilies. STR-PCR can also apply to non-deletion type carrier s family analysis. From 1994,prenatal diagnosis was performed in our lab for pregnant women at risk of having a DMD/ BMD baby. We established a series of genetic assays for the study,include:gender determination,multi- plex PCR analysis,mu
48、ltiplex fluorescence PCR capil- lary electrophoresis to detect the DMD gene deletion and duplication.Recently,We also developed the MLPA method,scanning for deletions and duplications of all the 79 DMD exons information. The diagnostic time is much fast than ever. MLPA for DMD diagnostic is the firs
49、t report method in China. Using a combined method,including:Fetus gen- der analysis by SRY gene amplification,multiplex PCR,multiplex fluorescence PCR capillary electropho- resis,MLPA and STR markers linkage analysis,we have achieved a combined diagnostic rate of 83%. Now we have developed a family-based strategy: (1)the genetic diagnosis of the individual with DMD / BMD, (2)the identification of potential carriers,and (3)possibly prenatal diagnosis. References 1Koenig M,Hoffman EP,Bertelson CJ,et al. Complete cloning of the Duchenne muscula
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