【作者】 钟敏；

【导师】 陆兵勋； 潘速跃；

【作者基本信息】 南方医科大学 ， 神经病学， 2009， 博士

【摘要】 DMD基因突变是假肥大型肌营养不良症的分子病理基础,其中大片段缺失约占所有突变类型的65%。依据阅读框学说,通常DMD基因移码突变导致Duchenne型肌营养不良（Duchenne muscular dystrophy,DMD）,而整码突变导致Becker型肌营养不良（Becker muscular dystrophy,BMD）。DMD基因缺失的机制甚为复杂,但在DMD基因发生断裂缺失后大都通过非同源末端连接进行修复。DMD基因缺失连接片段即为DMD基因断端重接形成的一段变异的DNA序列。对连接片段进行克隆和序列特点分析,可以为研究DMD基因缺失重组机制提供帮助;同时采用PCR技术扩增这段变异的DNA序列可以考虑用于缺失型DMD/BMD携带者检测和产前诊断。我们进行的研究工作分为三个部分:一、DMD基因缺失和5例中国人连接片段序列分析;二、利用连接片段进行缺失型DMD/BMD携带者检测;三、连接片段在缺失型DMD/BMD产前诊断的应用和评价。一、DMD基因缺失和5例中国人连接片段序列分析1.研究目的通过阅读框规则分析所选缺失型DMD/BMD患者基因型与表型的关系;通过进行DMD基因缺失连接片段测序,分析中国人连接片段序列的一些特点并探讨DMD基因缺失和修复的机制。2.方法研究对象:6例经证实的缺失型DMD/BMD患者。其中病例1、病例2、病例3、病例4为DMD,病例5、病例6为BMD,来自同一家系,故实际互相无血缘关系的研究对象为5例。抽提制备以上患者外周血基因组DNA。DMD基因缺失突变的阅读框检测:通过外显子PCR检测确定病例1为DMD基因第46～50外显子缺失,病例2为第51～55外显子缺失,病例3为第31～43外显子缺失,病例4为第45～54外显子缺失,相同家系的病例5、病例6为第3～5外显子缺失。采用在线程序对以上6例研究对象进行外显子缺失的阅读框检测,判断其缺失突变所致可读框改变类型。连接片段的克隆和测序:在研究对象DMD基因缺失断裂点所在的相应内含子上每间隔一定距离（一般为3 kb）设计1对引物,通过PCR步移的方法定位相应内含子上的断裂点位点。对5例研究对象内含子上断裂点位点成功定位后,分别在靠近其上下游断裂点处各设计1对配对引物,以直接对各例连接片段进行长片段PCR扩增,将扩增成功的PCR产物纯化后测序。连接片段的序列特点分析:对所测5例中国人连接片段5’端和3’端断裂点两侧的序列进行重复序列、基质附着区（matrix attachment regions,MARs）、回文序列、TTTAAA序列以及基因缺失后修复方式的分析。3.结果6例病例基因型和表型的关系:经检测病例1、病例2、病例3、病例4的缺失突变均属框外缺失（out-of-frame deletion）,为改变遗传信息可读框的移码突变,其相应的临床表型均为DMD。同一家系的病例5、病例6的缺失突变属框内缺失（in-frame deletion）,为缺失下游仍然保持正常可读框的整码突变,其相应的临床表型为BMD。5例中国人连接片段的序列特点:分析发现JUNCTION 1的5’端断裂点位于第45内含子LINE/L1序列内,邻近MAR,附近存在回文序列,其3’端断裂点邻近第50内含子的MAR;JUNCTION 2的5’端断裂点附近存在回文序列,其3’端断裂点位于第55内含子LINE/L1序列内;JUNCTION 3的5’端断裂点位于第30内含子LINE/L1序列内,附近存在回文序列;JUNCTION 4的5’端断裂点位于第44内含子LINE/L1序列内,邻近MAR,其3’端断裂点两旁存在回文序列;JUNCTION 5的5’端断裂点位于第2内含子SINE/Alu序列内,其3’端断裂点邻近第5内含子的MAR,附近有TTTAAA序列。5例DMD基因缺失相连接的序列均无广泛同源性。发现JUNCTION 1具有5 bp的微小同源序列连接断裂点两端;JUNCTION 2为平端连接,另外在其5’端断裂点上游39 bp位置发现一处A→G点突变;JUNCTION 3插入7 bp序列连接断裂点两端;JUNCTION 4具有4 bp的微小同源序列连接断裂点两端;JUNCTION 5插入26 bp序列并在连接点周围形成3个13 bp的重复连接断裂点两端,且在其5’端断裂点上游20 bp位置发现一处A→G点突变。4.结论6例假肥大型肌营养不良症病例基因型和表型的关系均符合阅读框规则,其中4例为out-of-frame缺失突变导致DMD,2例为in-frame缺失突变导致BMD。DMD基因的一些二级结构特点促进其断裂重组。5例中国人连接片段序列总体上具备容易导致DMD基因缺失重组的所有分子结构特点,包括重复序列、MARs、回文序列、TTTAAA序列等。并且5例中国人连接片段均采取非同源末端连接进行修复,其连接特点包括微小同源性、小的插入、重复、平端连接。二、利用连接片段进行缺失型DMD/BMD携带者检测1.研究目的依据DMD基因缺失后断端重接可形成的一段变异的DNA序列,提出一种利用连接片段进行缺失型DMD/BMD携带者检测的新方法。2.方法研究对象:2个DMD/BMD家系和1个散发病例家庭的女性亲属。其中家系1为一个BMD家系,家系中Ⅲ₁、Ⅲ₄为确诊的患者,先证者Ⅲ₁（病例5）已证实为第3～5外显子缺失并已克隆其连接片段JUNCTION 5,Ⅲ₁的母亲Ⅱ₂、Ⅲ₄的女儿Ⅳ4为肯定携带者,Ⅲ₁的妹妹Ⅲ₃为可能携带者;家系2为一个DMD家系,其中先证者Ⅲ₂（病例3）已证实为第31～43外显子缺失并已克隆其连接片段JUNCTION 3,Ⅲ₂的母亲Ⅱ₃为肯定携带者;病例4为一DMD散发病例,已证实为第45～54外显子缺失并已克隆其连接片段JUNCTION 4,其母亲待检测排除为携带者。抽提制备以上检测对象外周血基因组DNA。携带者检测:在连接片段序列已被成功测序的情况下,对扩增以上JUNCTION 5、JUNCTION 3、JUNCTION 4的引物进行重新设计减短PCR产物长度,以提高常规PCR扩增连接片段的成功率。然后以相应的新设计3对引物D4-F/R、D31-F/R2、D6-F/R分别对以上2个家系中女性待检测对象和病例4母亲可能存在的连接片段进行PCR扩增和测序,同时扩增患者的连接片段作对照。3.结果家系1检测结果:对家系1的3例女性Ⅲ₃、Ⅱ₂、Ⅳ4的连接片段PCR反应均为扩增出与患者Ⅲ₁、Ⅲ₄一致的阳性结果,同时比较Ⅲ₃、Ⅱ₂、Ⅳ₄与Ⅲ₁、Ⅲ₄的连接片段的测序结果无差异,诊断家系1的Ⅲ₃、Ⅱ₂、Ⅳ4均为BMD女性携带者。家系2检测结果:对家系2的1例女性Ⅱ₃的连接片段PCR反应为扩增出与患者Ⅲ₂一致的阳性结果,同时比较Ⅱ₃与Ⅲ₂的连接片段的测序结果无差异,诊断家系2的Ⅱ₃为DMD女性携带者。病例4的母亲检测结果:对病例4的母亲的连接片段PCR反应结果为扩增阴性,而对照的病例4扩增出阳性结果,可以排除病例4的母亲为DMD女性携带者。4.结论在对DMD/BMD先证者的连接片段的进行克隆和测序后,利用常规PCR技术直接检测待诊女性亲属是否带有连接片段,可以达到准确进行携带者检测的目的。通过测序分析表明相同家系中患者和女性携带者连接片段的序列完全一致,因此尽管不同家系患者的DMD基因缺失情况各异,但一般而言同一家系DMD基因缺失的基因型相同。三、连接片段在缺失型DMD/BMD产前诊断的应用和评价1.研究目的探讨利用连接片段进行缺失型DMD/BMD产前诊断的方法,通过实施评价其优势和不足。2.方法研究对象:在家系1的Ⅲ₃确诊为女性携带者之后,在知情同意的情况下将其作为实施产前诊断的对象。携带者Ⅲ₃共妊娠3次,其中Ⅳ₁、Ⅳ₂为先后两次人工流产的绒毛。Ⅳ₃为第三次妊娠待诊胎儿,分别在孕11周绒毛取样和孕20周取羊水标本进行产前诊断。抽提制备以上标本基因组DNA。人流绒毛的基因诊断方法:以新设计引物D4-F/R分别对以上2例人流绒毛Ⅳ₁、Ⅳ₂可能存在的连接片段进行PCR扩增和测序,同时扩增先证者Ⅲ₁的连接片段作为对照;以引物ex3-F/R分别检测Ⅳ₁、Ⅳ₂是否有相应外显子缺失;以扩增SPY基因的引物分别对Ⅳ₁、Ⅳ₂作性别鉴定。产前诊断方法:以新设计引物D4-F/R分别对携带者Ⅲ₃在不同孕期所取绒毛组织和羊水可能存在的连接片段进行PCR扩增和测序,并扩增先证者Ⅲ₁的连接片段作为对照;以引物ex3-F/R分别检测2次所取样品的DMD基因第3外显子,并以引物ex6-F/R扩增未缺失的第6外显子作为正常对照,判断是否有外显子缺失;以扩增SRY基因的引物分别对2次所取样品作性别鉴定。3.结果家系1的Ⅲ₃两次人流绒毛的基因诊断结果:对第一次人流绒毛Ⅳ₁的连接片段PCR反应为扩增出与先证者Ⅲ₁一致的阳性结果,对Ⅳ₁第3外显子的PCR检测结果为扩增阳性无缺失,对Ⅳ₁性别鉴定结果为女性。同时比较Ⅳ₁与本家系先证者Ⅲ₁的连接片段的测序结果无差异,诊断携带者Ⅲ₃第一次人流绒毛Ⅳ₁为BMD女性携带者。对第二次人流绒毛Ⅳ₂的连接片段PCR反应结果为扩增阴性,对Ⅳ₂第3外显子的PCR检测结果为扩增阳性无缺失,对Ⅳ₂性别鉴定结果为男性。诊断携带者Ⅲ₃第二次人流绒毛Ⅳ₂为正常男胎。家系1的Ⅲ₃第三次妊娠产前诊断结果:对孕11周所取绒毛组织基因组DNA的连接片段PCR反应结果为扩增出与先证者Ⅲ₁一致的阳性结果,对其第3外显子的PCR检测结果为扩增阳性无缺失,性别鉴定结果为男性。男性胎儿出现检测到DMD基因缺失连接片段而相应外显子无缺失的矛盾结果,提示所取的绒毛样品受到母体细胞污染。对孕20周羊水细胞基因组DNA的连接片段PCR反应结果为扩增出与先证者Ⅲ₁一致的阳性结果,对其第3外显子的PCR检测结果为扩增阴性（经验证第4外显子同为PCR扩增阴性结果）,存在相应的DMD基因缺失,性别鉴定结果为男性。同时比较该连接片段与本家系先证者Ⅲ₁的连接片段的测序结果无差异,诊断胎儿Ⅳ₃为BMD患病男胎并指导引产,取引产胎儿组织进行复查结果无误。4.结论在缺失型DMD/BMD产前诊断上通过PCR扩增连接片段和检测是否有相应的外显子缺失,可以准确判断男性胎儿是否为患病胎儿,并能有效鉴别检测标本是否受到母体细胞污染。不足之处是比较繁琐和耗时,且仅能应用于缺失型DMD/BMD。总结:1.6例缺失型DMD/BMD研究对象在DNA水平均符合阅读框规则。2.报道了迄今为止数量最多的东方亚洲人DMD基因缺失连接片段序列资料,共完成5例中国人连接片段的测序。3.DMD基因的一些二级结构特点促进其断裂重组。5例中国人连接片段序列总体上具备容易导致DMD基因缺失重组的所有分子结构特点,包括重复序列、MARs、回文序列、TTTAAA序列等。4.5例中国人连接片段均为采取非同源末端连接进行修复,其连接特点包括微小同源性、小的插入、重复、平端连接。5.首次对相同DMD/BMD家系中多个患者和携带者的DMD基因缺失连接片段进行测序分析。6.发现尽管不同家系患者的DMD基因缺失情况各异,但一般而言同一家系DMD基因缺失的基因型相同。7.成功利用常规PCR扩增连接片段的方法对3个不同缺失型DMD/BMD家庭进行携带者检测。8.该方法与目前常用携带者检测方法的主要区别是不对DMD基因进行定量分析,只要对女性被检者检测出与先证者一致的连接片段即可确认为携带者,方法直观、准确。9.首次将连接片段应用于1例BMD女性携带者产前诊断,实现了利用连接片段进行缺失型DMD/BMD产前诊断的设想。10.连接片段应用于DMD/BMD基因诊断的技术缺点是首先需要分析先证者的基因型,比较繁琐和耗时,且仅能应用于缺失型DMD/BMD。创新点:1.克隆了5例新的DMD基因缺失突变序列。2.首次将基于常规PCR技术扩增连接片段的方法应用于缺失型DMD/BMD携带者检测和产前诊断。3.突破了以往认为常规PCR技术不能检测DMD/BMD携带者的观点。4.在缺失型DMD/BMD产前诊断上通过PCR扩增连接片段和检测是否有相应的外显子缺失,可以准确判断男性胎儿是否为患病胎儿,并能有效鉴别检测标本是否受到母体细胞污染。5.该方法对检测样品需求量小,具有应用于植入前遗传学诊断可行性。更多还原

【Abstract】 DMD gene mutation is the molecular pathological basis for pseudohypertrophic muscular dystrophy.The large gene fragment deletion accounts for～65%in all the mutants.According to the reading-frame hypothesis,the frameshift mutations of DMD gene generally result in Duchenne muscular dystrophy（DMD）,whereas the codon mutations result in Becker muscular dystrophy（BMD）.Although the mechanism of DMD gene deletion is very complicated,it can be found that DMD gene with breaks and deletions are almost repaired by non-homologous end joining, which results in a new unique DNA sequence—DMD gene deletion junction fragment by the illegitimate recombination of the two ends of breakpoints in each case.Cloning and analyzing the sequence features of the junction fragments is useful for studying the mechanism of DMD gene deletion and recombination.We have also considered that using the PCR technique to amplify this unique DNA sequence could be apply to deletional DMD/BMD carrier detection and prenatal diagnosis.This study has 3 chapters:1.Analysis of DMD gene deletion and the sequences of 5 Chinese junction fragments;2.Using the junction fragment for deletional DMD/BMD carrier detection;3.Application and evaluation of the junction fragment for deletional DMD/BMD prenatal diagnosis.1.Analysis of DMD gene deletion and the sequences of 5 Chinese junction fragments （1）ObjectiveUsing the reading-frame rule to analyze the relationship between the genotype and the phenotype of the choosed deletional DMD/BMD patients.Sequencing the Chinese DMD gene deletion junction fragments to analyze some features of them and discuss the mechanism of DMD gene deletion and repair.（2）MethodsStudy objects:6 definite deletional DMD/BMD patients.Case 1,case 2,case 3, case 4 were affected by DMD.Case 5 and case 6 from a same family were affected by BMD.So actually there were 5 cases that had no blood relationship each other. The peripheral blood genomic DNA of these patients was extracted and prepared.Analysis of DMD gene deletion mutations by the reading-frame rule:Using exon primers for PCR detection,case 1 was substantiated with exons 46-50 deletion of DMD gene,and case 2 with exons 51-55 deletion,case 3 with exons 31-43 deletion, case 4 with exons 45-54 deletion,Case 5 and case 6 from the same family with exons 3-5 deletion.Then the 6 cases were analyzed by on line reading-frame program to know their changed types of open reading frame caused by the exons deletion.Cloning and sequencing of the junction fragments:We first designed the primer at intervals（often 3 kb sequence）in the corresponding introns that the DMD gene deletion breakpoints of the DMD/BMD cases were located in.After the breakpoints in introns of the 5 cases were localized by the PCR-based genome-walking method successfully,we designed 1 pair of matched-pair primer close to the upstream and the downstream breakpoints respectively to be used in long fragment PCR amplification of each junction fragment.Sequencing after purifying successfully PCR productions.Analysis of the sequence features of the junction fragments:The sequences both sides of 5’ and 3’ breakpoints of 5 forementioned Chinese junction fragments were analyzed by means of repetitive sequence,matrix attachment regions（MARs）, palindromic sequence,the sequence TTTAAA and the repair means after gene deletion.（3）ResultsRelationship between the genotype and the phenotype of the 6 cases:Through the detection,the deletion mutations of case 1,case 2,case 3 and case 4 all belong to out-of-frame deletion which is the frameshift mutation changing the genetic information of open reading frame.The corresponding clinical phenotype is DMD. The deletion mutations of case 5 and case 6 from the same family belong to in-frame deletion which is the codon mutation maintaining open reading frame correct downstream the deletion.The corresponding clinical phenotype is BMD.Sequence features of 5 Chinese junction fragments:It was found that the 5’ breakpoint of JUNCTION 1 was located in LINE/L1 element of intron 45 and close to a MAR,with a palindromic sequence nearby.It’s 3’ breakpoint was close to a MAR of intron 50.The 5’ breakpoint of JUNCTION 2 had a palindromic sequence nearby.It’s 3’ breakpoint was located in LINE/L1 element of intron 55.The 5’ breakpoint of JUNCTION 3 was located in LINE/L1 element of intron 30,with a palindromic sequence nearby.The 5’ breakpoint of JUNCTION 4 was located in LINE/L1 element of intron 44 and close to a MAR.It’s 3’ breakpoint had a palindromic sequence beside.The 5’ breakpoint of JUNCTION 5 was located in SINE/Alu element of intron 2.It’s 3’ breakpoint was close to a MAR of intron 5,with the sequence TTTAAA nearby.There wasn’t extensive homogeneity in gene deletion conjuncted sequence in the 5 cases.A 5 bp microhomologous sequence was found in the joint of JUNCTION 1. The blunt end ligation was found in the joint of JUNCTION 2,with an A→G point mutation at 39 bp site upstream it’s 5’ breakpoint.A 7 bp inserted sequence was found in the joint of JUNCTION 3.A 4 bp microhomologous sequence was found in the joint of JUNCTION 4.A 26 bp inserted sequence was found in the joint of JUNCTION 5 which formed three 13 bp duplications around it,with an A→G point mutation at 20 bp site upstream it’s 5’ breakpoint.（4）ConclusionThe relationship between the genotype and the phenotype of the 6 pseudohypertrophic muscular dystrophy cases all fit with the reading-frame rule.4 cases of them are out-of-frame deletions leading to DMD.2 cases of them are in-frame deletions leading to BMD.Some secondary structures of DMD gene facilitate gene breakage and recombination.Overall,the sequences of 5 Chinese junction fragments of our study have all the molecular features including repeated sequence,MARs,palindromic sequence,the sequence TTTAAA,etc,which cause DMD gene deletion and recombination easily.Moreover,5 Chinese junction fragments are all repaired by non-homologous end joining whose characteristic junctions showing microhomologies,small insertions,duplications,or blunt end ligation.2.Using the junction fragment for deletional DMD/BMD carrier detection（1）ObjectiveOn the basis of DMD gene deletion junction fragment is the unique DNA sequence resulted from illegitimate recombination after the gene deletion,a novel approach is presented here for the detection of deletional DMD/BMD carriers with the junction fragments.（2）MethodsStudy objects:The female relations of 2 DMD/BMD families and a sporadic case’s family.Family 1 was a BMD family.In this familyⅢ₁ andⅢ₄ were definite patients.The probandⅢ₁（Case 5）had been substantiated with exons 3-5 deletion and its junction fragment（JUNCTION 5）had been cloned.Ⅲ₁’s motherⅡ₂ and Ⅲ₄’s daughterⅣ₄ were definite carriers.Ⅲ₁’s younger sisterⅢ₃ was a possible carrier.Family 2 was a DMD family.In this family the probandⅢ₂（Case 3）had been substantiated with exons 31-43 deletion and its junction fragment（JUNCTION 3）had been cloned.Ⅲ₂’s motherⅡ₃ was a definite carrier.Case 4 was a DMD sporadic case that had been substantiated with exons 45-54 deletion and its junction fragment（JUNCTION 4）had been cloned.His mother was to be excluded as a carrier. The peripheral blood genomic DNA of them was extracted and prepared.Carriers detection:On the basis of successful sequencing of the junction fragments,we redesigned the primers that amplified above JUNCTION 5, JUNCTION 3,JUNCTION 4 to elevate the achievement ratio of routine PCR amplifying the junction fragments by cutting short the PCR products length.Then 3 pairs of redesigned primer D4-F/R,D31-F/R2,D6-F/R were used to PCR amplify and sequence the possible junction fragments of the female objects of above 2 families and case 4’s mother respectively.At the same time the junction fragments of the patients were amplified as control.（3）ResultsResults of family 1:The PCR amplifications of the junction fragments of 3 femalesⅢ₃,Ⅱ₂,Ⅳ₄ of family 1 were the same positive results as those of patientsⅢ₁ andⅢ₄.Meanwhile the sequences of the junction fragments ofⅢ₃,Ⅱ₂,Ⅳ₄ andⅢ₁,Ⅲ₄ had no difference by sequencing comparison.SoⅢ₃,Ⅱ₂,Ⅳ₄ of family 1 were all diagnosed as female carriers of BMD.Results of family 2:The PCR amplification of the junction fragment of a femaleⅡ₃ of family 2 was the same positive result as that of patientⅢ₂.Meanwhile the sequences of the junction fragments ofⅡ₃ andⅢ₂ had no difference by sequencing comparison.SoⅡ₃ of family 2 was diagnosed as female carrier of DMD.Results of case 4’s mother:The PCR amplification of the junction fragment of case 4’s mother was negative result.But the control of case 4 was positive result.So case 4’s mother was excluded as female carrier of DMD.（4）ConclusionAfter the junction fragment of the DMD/BMD proband was cloned and sequenced,using the routine PCR technique to detect the female relation to be diagnosed having the junction fragment or not directly can achieve the aim of accurate carrier detection.The sequencing results indicate that the junction fragments of the patients and the carriers from the same families are identical,so generally the same family has the same genotype of DMD gene deletion despite the patients from the different families having the different deletions.3.Application and evaluation of the junction fragment for deletional DMD/BMD prenatal diagnosis（1）ObjectiveTo study the approach of using the junction fragment for deletional DMD/BMD prenatal diagnosis.It’s advantage and disadvantage has been evaluated through the practice.（2）MethodsStudy objects:AfterⅢ₃ of family 1 was diagnosed as female carrier,she was choosed as the study object of practising prenatal diagnosis under her agreement. CarrierⅢ₃ was gravida 3.Ⅳ₁ andⅣ₂ were 2 chorionic villi from her successive artificial abortion.Ⅳ₃ was the fetus of tertigravida to be diagnosed.The chorionic villi at 11 weeks’ gestation and the amniotic fluid at 20 weeks’ gestation were taken respectively as the samples of prenatal diagnosis.The genomic DNA of these samples was extracted and prepared.The methods of gene diagnosis of the chorionic villi from artificial abortion:The redesigned primer D4-F/R was used to PCR amplify and sequence the possible junction fragments ofⅣ₁ andⅣ₂ respectively.At the same time the junction fragment of probandⅢ₁ was amplified as control.The primer ex3-F/R was used to identifyⅣ₁ andⅣ₂ with corresponding exons deletion or not respectively.The primer of amplifying SRY gene was used to diagnose the sex ofⅣ₁ andⅣ₂ respectively.The methods of prenatal diagnosis:The redesigned primer D4-F/R was used to PCR amplify and sequence the possible junction fragments of the samples of the chorionic villi and the amniotic fluid at different gestation respectively.At the same time the junction fragment of probandⅢ₁ was amplified as control.The primer ex3-F/R was used to amplify exon 3 of the 2 samples to identify them with exons deletion or not respectively,with the primer ex6-F/R being used to amplify the undeleted exon 6 as normal control.The primer of amplifying SRY gene was used to diagnose the sex of the 2 samples respectively.（3）ResultsResults of gene diagnosis of 2 chorionic villi fromⅢ₃ ’s artificial abortion of family 1:The PCR amplification of the junction fragment of the first chorionic villiⅣ₁ was the same positive result as that of probandⅢ₁.The PCR amplification of exon 3 ofⅣ₁ was positive result without deletion.The sex diagnosis ofⅣ₁ was female.Meanwhile the sequences of the junction fragments ofⅣ₁ and probandⅢ₁ of the family had no difference by sequencing comparison.So the chorionic villiⅣ₁ from carrierⅢ₃’s first artificial abortion was diagnosed as female carrier of BMD.The PCR amplification of the junction fragment of the second chorionic villiⅣ₂ was negative result.The PCR amplification of exon 3 ofⅣ₂ was positive result without deletion.The sex diagnosis ofⅣ₂ was male.So the chorionic villiⅣ₂ from carrierⅢ₃’s second artificial abortion was diagnosed as normal male fetus. Results of prenatal diagnosis of tertigravidaⅢ₃ of family 1:The PCR amplification of the junction fragment of the chorionic villi sample genomic DNA prepared at 11 weeks’ gestation was the same positive result as that of probandⅢ₁. The PCR amplification of it’s exon 3 was positive result without deletion.It’s sex diagnosis was male.The contradictory outcome of DMD gene deletion junction fragment being detect but without corresponding exons deletion in a male fetus indicated that the chorionic villi sample had been contaminated by maternal cell.The PCR amplification of the junction fragment of the amniotic cell genomic DNA prepared at 20 weeks’ gestation was the same positive result as that of probandⅢ₁.The PCR amplification of it’s exon 3 was negative result（It was checked by exon 4 which was also negative amplification）,with corresponding exons deletion.It’s sex diagnosis was male.Meanwhile the sequence of it’s junction fragment and that of probandⅢ₁ of the family had no difference by sequencing comparison.So fetusⅣ₃ was diagnosed as diseased male fetus with BMD and was instructed in induced abortion.The result was rechecked without mistake by the fetus tissue from the induced abortion.（4）ConclusionBy PCR amplifying the junction fragment and detecting the corresponding exons with deletion or not for deletional DMD/BMD prenatal diagnosis,the male fetus can be diagnosed accurately as diseased fetus or not.As well as the study sample can be identified effectively being contaminated by maternal cell or not.The disadvantage of this approach is laborious and time-consuming,and can only be applied to deletional DMD/BMD.Summarization（1）The 6 deletional DMD/BMD cases all fit with the reading-frame rule at the DNA level. （2）Our study has fulfilled the sequencing of 5 Chinese junction fragments, reporting the largest sequence data of DMD gene deletion junction fragments of east Asian people up to now.（3）Some secondary structures of DMD gene facilitate gene breakage and recombination.Overall,the sequences of the 5 Chinese junction fragments have all the molecular features including repeated sequence,MARs,palindromic sequence, the sequence TTTAAA,etc,which cause DMD gene deletion and recombination easily.（4）The 5 Chinese junction fragments are all repaired by non-homologous end joining whose characteristic junctions showing microhomologies,small insertions, duplications,or blunt end ligation.（5）The DMD gene deletion junction fragments of several patients and carriers from the same DMD/BMD families have been sequenced and analyzed for the first time.（6）Our study discovers that generally the same family has the same genotype of DMD gene deletion despite the patients from the different families having the different deletions.（7）Our study has used routine PCR amplifying the junction fragment for the carriers detection of 3 different deletional DMD/BMD families successfully.（8）The main difference between this approach and the common approaches of carrier detection at present is that it doesn’t need quantitative analysis of DMD gene. The female object can be diagnosed as a carrier as long as she was detected in having the same junction fragment as the proband,with direct-viewing and accurateness.（9）Our study has applied the junction fragment to prenatal diagnosis for a female carrier of BMD for the first time,realizing the presumption that the junction fragment could be applied to deletional DMD/BMD prenatal diagnosis. （10）The disadvantage of the approach of using the junction fragment for gene diagnosis of DMD/BMD is that the genotype of the proband need to be investigated first,which is laborious and time-consuming,and can only be applied to deletional DMD/BMD.Innovation（1）5 new sequences of DMD gene deletion mutation have been cloned.（2）The approach based on routine PCR amplifying the junction fragment has been used for deletional DMD/BMD carrier detection and prenatal diagnosis for the first time.（3）The previous viewpoint that the routine PCR technique can’t detect the female carriers of DMD/BMD has been broken through.（4）By PCR amplifying the junction fragment and detecting the corresponding exons with deletion or not for deletional DMD/BMD prenatal diagnosis,the male fetus can be diagnosed accurately as diseased fetus or not.As well as the study sample can be identified effectively being contaminated by maternal cell or not.（5）The approach has the feasibility of being applied to preimplantation genetic diagnosis,with a little check sample needed.更多还原