摘要
假肥大型肌营养不良症(Duchenne Muscular Dystrophy/Becker's Muscular Dystrophy, DMD/BMD)是最常见的X-连锁隐性致死性遗传病之一,由Duchenne等(1868年)首先报道,故得其名。本病的群体发病率高达1/3500活产男婴,是一种预后不良的常见的原发性肌肉疾病。典型的临床特征是进行性肌萎缩、肌无力伴小腿腓肠肌的假性肥大,通常累及青少年男性,一般在12岁以前丧失站立和行走的能力,最后因心肌以及呼吸肌无力而多于20岁前死于心力衰竭或呼吸衰竭。本病严重影响了青少年男性的健康成长,同时也给家庭和社会带来了沉重的精神和经济负担。
本病基因定位在人类X染色体短臂Xp21上,DMD基因突变是本病各型临床亚型患者发病的共同的分子遗传学基础。DMD基因突变的主要形式有三类：缺失型突变、重复型突变和点突变,前两种突变约占了全部基因突变的70%,点突变约占30%。而所有的突变中约70%遗传自母亲,约30%没有家族史,为新发突变。
国内外至今尚缺乏对于本病有效的治疗措施,因此,对先证者进行确诊、对携带者进行产前诊断以杜绝患儿出生的出生缺陷干预措施仍然是目前国内外假肥大型肌营养不良症遗传优生关键之所在。
在基因诊断技术开展以前,本病的临床诊断除依靠典型的症状和体征外,还需要结合肌电图、酶生化检查和肌肉活检等辅助性检查,其中肌电图显示肌源性损害及酶生化检查发现肌酶活性显著增高是较可靠的临床诊断依据。但上述血清酶的增加特异性并不高,并且存在假阳性和假阴性,而且不能应用在胎儿的产前诊断上,导致在临床上应用受到一定的限制。
随着DNA研究技术的发展,各种基因诊断的手段也被应用到假肥大型肌营养不良症的检测中来。国外DMD基因诊断的主要手段有：Southern印迹杂交技术、DMD基因多重PCR技术、短串联重复序列PCR技术及反转录PCR技术。Chamberlain等设计了9对引物的多重PCR,可检出80%的DMD基因缺失型患者；Beggs等增设了另外9对引物多重PCR,这18对引物总共可以检出98%的DMD基因缺失型患者。但这种方法的局限是不能检测出基因缺失型和基因重复型的杂合子携带者。Prior等最先应用定量PCR原理诊断了杂合子携带者。短串联重复序列PCR技术也被用于非缺失型家系的基因连锁分析。
DMD产前基因诊断是一个比较复杂的难题,我们以前主要采用性别诊断、多重PCR缺失突变分析、STR单体型连锁分析等方法进行。这些方法有一定的局限性,不能很好地适应产前诊断的需要。单一的性别诊断会导致正常的男性胎儿被淘汰掉。由于存在高达11%的基因内交换率,理论上STR单体型连锁分析结果具有可高达11%假阳性或假阴性率,因此,这种单体型连锁分析诊断结果只是一种概率性诊断,有时也因缺乏先证者和杂合信息或家系过小而无法展开连锁分析。多重PCR方法所得到的遗传信息量不够大,容易导致漏诊,而且不能检测出重复突变,也不能检测出杂合子携带者。鉴于上述缺陷,建立各种DMD基因杂合子非概率性确诊技术就成为最终解决DMD杂合子携带者诊断的关键,是DMD产前基因诊断的前提条件。
针对DMD基因三大类突变类型,我们拟建立高效、省时的针对DMD基因全部79个外显子的缺失型、重复型和点突变型基因突变的检测方法,对患者以及携带者作出正确的诊断,并将其应用于产前诊断；探索产前诊断时机前移的可行性,以达到早期诊断、杜绝患胎出生、优生优育的目的。
本研究应用多重连接依赖性探针扩增(Multiplex Ligation dependent ProbeAmplification, MLPA)等方法对DMD先证者及其母亲进行检测,筛选出准备生育下一胎的DMD携带者进行产前基因诊断,检测DMD全部79个外显子的缺失型与重复型突变,建立准确的DMD患者和杂合子携带者的基因诊断技术；建立改良的96孔板一步全外显子测序方法,用于点突变型DMD基因突变的检测和产前诊断；摸索高分辨溶解曲线(High Resolution Melting, HRM)用于DMD突变的初筛方法；摸索单卵裂球的全基因组扩增方法,为下一步开展胚胎种植前遗传学诊断和筛查(PGD/PGS)、囊胚优化提供实验准备；探讨女性DMD患者的发病机理；建立羊水来源细胞诱导多能干细胞系(iPSCs),为DMD的基因治疗研究和疾病模型建立提供良好的研究基础。
第一章缺失型和重复型DMD基因突变诊断技术的建立及其在产期诊断中的应用
研究目的
本部分的研究目的在于建立更准确、高效、省时的基因诊断方法应用于缺失型和重复型DMD基因突变的诊断和产前诊断。
建立适宜的实验方法筛查DMD全部79个外显子的缺失与重复突变。建立准确的DMD患者和杂合子携带者的基因诊断技术。在前期研究基础上,筛选出准备生育下一胎的DMD家系进行产前基因诊断,避免以往正常的男性胎儿被无辜地淘汰掉的情况发生,建立一套临床可行的DMD产前基因诊断方法。
本章研究还探讨早期产前诊断的方法与时机,尽量把检测时机提早,从中期羊水检查提早到早期绒毛的产前诊断,便于终止妊娠手术的提前,减轻孕妇的痛苦,为出生缺陷的早期干预提供实验基础。
对单卵裂球进行DNA全基因组扩增,摸索可用于DMD胚胎种植前诊断的实验方法,为下一步开展胚胎种植前诊断和胚胎种植前遗传学筛查、囊胚优化提供实验准备。
研究方法
资料来源：
1、病例主要来源于广州医学院第三附属医院、中山大学附属第一医院、广州市儿童医院和南方医科大学南方医院,先证者均具有典型的DMD临床表现,经血清肌酶、肌电图或肌组织活检等检查,排除了其他神经肌肉系统遗传病；
2、携带者妊娠期抽取羊水组织或绒毛组织；
3、知情同意废弃的卵裂期单细胞及已知基因型DMD患者的单淋巴细胞。
实验方法：
1、应用MLPA法对先证者进行检测：采用SALSA probe mix P034和P035(MRC Holland)经变性、杂交、连接反应、PCR反应和ABI3100遗传分析仪毛细管电泳对产物进行分析,检测DMD基因79个外显子缺失型以及重复型基因突变。
2、筛选出155个有再生育要求,并且已明确携带了DMD基因杂合缺失或基因杂合重复的DMD携带者,对其风险胎儿进行产前诊断：其中,对148例携带者孕妇于妊娠16周以后行羊膜腔穿刺术以获得羊水样本；7例携带者孕妇于妊娠9-12周左右行绒毛膜穿刺术抽取绒毛组织。
3、绒毛组织检查时同时抽取携带者孕妇血液,对绒毛样本和母体样本进行DNA-STR分型。
4、用RFPLI-g Midi对单细胞全基因组扩增(WGA)—多重置换扩增(MDA),并用PCR及DMD-STR位点扩增进行验证。
研究结果
1、DMD先证者及其母亲的基因诊断结果：对1119例临床疑似DMD患者进行了基因诊断,检测出缺失型和重复型DMD患者共725例,其中DMD基因缺失突变607例,占54%(607/1119),DMD基因重复突变118例,占11%(118/1119),未发现缺失和重复的394例,占35%(394/1119)。
2、DMD先证者缺失突变和重复突变的突变来源比较：在607例基因缺失突变患者中,342例母亲为缺失突变携带者,265例为新发突变,突变率为43.66%(265/607)；在118例基因重复突变的患者中,96例母亲为重复突变携带者,22例为新发突变,突变率为18.64%(22/118)。经卡方检验,Pearson Chi-Square=25.846P<0.001,两种突变类型的来源有显著性差异。
3、MLPA法与荧光多重PCR方法的比较：对55例MLPA方法检测DMD基因缺失或重复的DNA样本用荧光多重PCR (fmPCR)方法进行检测,可见55例中有4例用fmPCR检不出的,而用MLPA方法能获得79个外显子全部信息,对基因的缺失与重复的检测没有出现漏诊。
4、缺失型和重复型DMD携带者产前诊断
(1)产前诊断的结果：我们总共对155例诊断为缺失型或重复型携带者孕妇进行了产前诊断,其中148例抽取中期妊娠羊水、7例抽取早期妊娠绒毛组织。检出DMD患胎27例,占17%(27/155)；胎儿DMD携带者28例,占18%(28/155)；正常胎儿100例,占65%(100/155)。对DMD患胎给予终止妊娠,正常胎儿及DMD携带者胎儿给予继续妊娠的处理。
(2)胎儿的性别情况：155例产前诊断胎儿中,男胎72例,女胎83例。在72例男胎中,27例为患胎,占全部男胎的38%(27/72),45例为正常男胎,占63%(45/72)；在83例女胎中,28例胎儿为携带者,占女胎的34%(28/83)；55例为正常女胎,占66%(55/83)。
(3)DMD基因突变的类型：在检出的27例患胎中,DMD外显子缺失突变22例(14.19%,22/155)；外显子重复突变5例(3.23%,5/155)；在检出的28例胎儿DMD基因携带者中,杂合缺失突变25例(16.13%,25/155),杂合重复突变3例(1.94%,3/155)；正常胎儿100例,占64.52%。
(4)DMD外显子累计被缺失与重复突变涉及的次数：本研究中DMD基因缺失和重复突变主要分布在外显子45-52之间,为突变热点区域,其中外显子49突变发生次数最高,共22次；在本研究中外显子1和2未发现突变,外显子56～79之间的突变发生次数较低,绝大部分只涉及1次；外显子73涉及过2次。
5、早期妊娠绒毛组织DMD产前基因诊断：对7例携带者进行了孕早期的DMD产前诊断：抽取早孕绒毛组织,用MLPA方法进行全外显子的检测,并对样本及母体进行DNA-STR分型以排除母源性污染。基因诊断结果：1例为DMDexon48-50基因缺失突变、胎儿DMD基因诊断明确为患儿,孕妇选择终止妊娠；1例为DMD exon16-42杂合重复突变,胎儿DMD基因诊断明确为DMD携带者,给予继续妊娠的处理；其余5例胎儿均正常。
6、对8个单胚胎细胞进行了全基因组多重置换扩增(WGA-MDA),并用PCR方法对DYSII、DMD外显子50、外显子49、外显子12和外显子17对MDA的扩增效果进行验证：并用DMD44、49、50STR、DYSII STR及3'CASTR进行验证,均可得到预期结果。
研究结论
1、MLPA方法是目前为止检测DMD缺失型和重复型基因突变的最有效的方法,能鉴别突变是缺失型还是重复型,也能区分患者与携带者,可用于该类型患者的诊断及携带者的产前诊断。
2、采用MLPA方法结合DNA-STR分型技术对绒毛组织进行检测可用于DMD的孕早期诊断,有利于早期进行出生缺陷干预。
3、本研究DMD基因突变主要分布在外显子45-52之间,其中外显子49突变发生次数最高,为突变热点区域。
4、DMD外显子缺失突变和重复突变的突变率有显著性差异,后者多遗传自母亲,新发突变较少。
5、已建立了单卵裂球全基因组扩增-多重置换扩增实验方法,并通过了DMD外显子的PCR和STR对其扩增效果的验证,为下一步开展胚胎种植前诊断和胚胎种植前遗传学筛查、囊胚优化提供实验准备。
第二章点突变型DMD基因诊断与筛查技术的建立及应用研究
研究目的
通过上一章所建立的诊断技术流程,已经可以解决基因的缺失突变和基因重复突变这两类的患者诊断、携带者诊断及其产前诊断问题,也就是解决了约70%的DMD诊断问题。而剩下的约30%患者是由DMD基因的点突变引起,MLPA无法检测点突变。由于DMD基因的庞大,目前我国还没有医疗机构能常规地对DMD基因点突变的患者进行检测,因而也没办法对点突变的家系进行产前诊断。
针对DMD如此庞大的基因,本研究拟建立更高效的全外显子测序方法检测其点突变,在一块96孔板上加样,经一次的测序流程即可达到对全部79个外显子的测序。通过本研究,可为目前临床得不到常规基因诊断的点突变患者及携带者提供确诊方法、进行产前诊断,最终可达到把DMD的基因诊断率从现时的70%至少提高到90%以上,将大大减少DMD患儿的出生。
本研究还摸索用高分辨溶解曲线(HRM)方法对DMD点突变进行筛查,试图寻找一种可以用于点突变初筛的方法,以图降低实验成本。
在本研究的另一部分,我们对DMD单个外显子缺失情况进行了深入的研究,用基因测序结合PCR方法对MLPA判读为单个外显子缺失的DMD患者进行验证,鉴别单外显子是真正缺失还是点突变,避免由此引出的误诊,从而达到准确诊断的目的,并为基因修复治疗提供理论依据。
研究方法
资料来源：病例主要来源本院,先证者具有典型的临床表现,经血清肌酶、肌电图或肌活检证实,并排除其它类型的神经肌肉系统遗传病,经MLPA证实不存在DMD基因外显子的缺失和重复。
实验方法：
1、采用Qiagen方法提取DMD患者外周血样本基因组DNA；
2、用MLPA方法排除先证者存在DMD基因外显子的缺失和重复；
3、用改良的96孔板全外显子测序方法对DMD基因全部79个外显子进行测序：经PCR、PCR产物纯化、测序反应、测序PCR产物纯化、ABI3100遗传分析仪进行毛细管电泳、用GeneMapper ID v3.1软件和Mutation Surveyor软件进行结果分析等步骤获得测序结果。
4、采用LightScanner HRM32分析仪进行HRM检测,对某些未知点突变和已知点突变样本进行检测。
5、对MLPA法判断为单个外显子缺失的患者进行PCR及测序验证。
研究结果
1、用改良的96孔板全外显子测序方法对DMD基因全部79个外显子进行分析：选择临床诊断为DMD患者,并且排除其他神经肌肉疾病、且经MLPA排除DMD缺失突变与重复突变的16例样本进行了测序,本研究发现的点突变有：单碱基置换13例、单碱基缺失2例、2个碱基缺失1例,5个碱基插入1例,该例碱基的插入和三例碱基缺失导致了DMD的移码突变。
2、应用测序结合PCR方法对MLPA诊断为单个外显子缺失的DMD患者进行验证,以鉴别真缺失还是点突变：37例MLPA法判断为单外显子缺失的样本中30例确实为DMD基因外显子的缺失,占单个外显子缺失中的81.08%；而另外7例经PCR法验证未发现缺失,经测序确证为存在外显子的点突变,占单个外显子缺失中的18.92%。
3、HRM筛查DMD点突变方法的摸索
(1)引物最佳退火温度的优化：设置Tm58℃/59℃/60℃/61℃/62℃/63℃/64℃/65℃,选择61℃为最佳退火温度。
(2)DMD患者HRM结果：对10例DMD点突变的样本进行了HRM分析,其中7例可以出现突变曲线,其余3例未能出现预期的结果。
4、对一例携带有DMD外显子54：c.7874A>G(家系156C)的点突变携带者孕妇进行了羊水产前诊断,胎儿为男性,结果为正常。
研究结论
1、改良的96孔板全外显子测序方法可通过一块96孔板在一次实验中完成对79个外显子的全测序,是对点突变型DMD基因突变的高效可靠的检测方法。测序技术可用于DMD点突变携带者产前诊断。
2、高分辨溶解曲线(HRM)能快速地对一些稀有突变基因型进行初筛检测,但本研究不能分辨出300bp长度以上的扩增子中一个碱基的变化,对于扩增子为300bp以上的外显子需重新进行引物设计、PCR条件优化。
3、经MLPA检测DMD基因为单个外显子缺失时,应联合应用PCR和基因测序技术进行鉴别,以区分真缺失还是点突变,提高诊断的准确率。
第三章女性DMD发病机制研究及羊水iPS细胞系建立
研究目的
DMD/BMD为X染色体隐性遗传病,理论上发病者为男性,所以女性发病者多被误诊为与其症状相似的常染色体隐性遗传的肢带型肌营养不良,近年来研究显示女性患者的发病机制可能涉及X染色体失活等表观遗传调控,因此,研究女性发病者的分子机理具有较为重要的临床价值和科学意义。本研究利用测序、染色体核型分析等方法、并采用Affymetrix CytoScan HD高分辨率基因芯片对女性DMD患者进行全基因组检测,分析其是否微小缺失／重复,采用甲基化特异的PCR检测X染色体失活状态,研究其与女性DMD发病的相关性。
DMD至今无特异性治疗,只能对症治疗和支持治疗。寻找有效的方法治疗DMD一直是神经病学界研究的热点话题。诱导性多能干细胞(iPS)以其较好的免疫相容性被认为是可以作为治疗各种疾病的潜在来源,也是疾病机理研究的理想模型。羊水中细胞成分众多,目前认为羊水细胞是来源于胎儿和羊膜的异质细胞群体,由不同来源的多种细胞组成。羊水细胞具有良好多能性,可被诱导分化为各个胚层的细胞类型,本研究拟利用OCT4、KLF4两因子法诱导羊水细胞重编程,获得iPSCs,为DMD的干细胞治疗和疾病模型建立提供良好的研究基础。
研究方法
资料来源：
1、选择在广州医学院第三附属医院就诊的4名女性DMD患者；
2、羊水细胞：收集我院产前诊断中心具有羊水穿刺指证的1名孕妇20孕周,产前诊断检测后废弃的羊水细胞,经捐赠夫妇知情同意及伦理委员会同意用于本实验。
实验方法：
1、用Qiagen方法提取女性DMD患者外周血样本基因组DNA,按照Affymtrix公司CytoScan HD芯片操作规程进行操作及质检；
2、X染色体失活状态检测：基因组DNA经过硫酸盐处理后采用甲基化特异的引物进行扩增,产物进行毛细管电泳分析。
3、iPS细胞制备：经羊水细胞收集传代、羊水细胞感染、hAFDC-iPSCs原代培养、hAFDC-iPSCs传代、RT-PCR检测多能性基因、碱性磷酸酶(AP)检测、免疫荧光染色、体外分化能力检测、体内分化潜能的检测及染色体核型分析等主要步骤,制备iPS细胞。
研究结果
1、DMD基因双突变是女性患者发病机理之一：对一例女性DMD患者测序发现存在着DMD外显子10：c.1034A>G和DMD外显子21：c.2645G>A两处单碱基置换突变。估计这两处突变发生在不同的X染色体上,为杂合重复突变,导致女性发病。
2、染色体核型异常导致女性DMD发生：对一例女性DMD患者进行了染色体核型分析,其核型是：46X, iX (q10),即她的两条X染色体中的一条为X长臂等臂染色体,这一条染色体的短臂是丢失的,位于短臂上的DMD基因也因此丢失,而另一条正常的X染色体上DMD外显子46-47发生了缺失突变,导致了这例女孩的发病。
3、高分辨率基因芯片分析女性患者DMD基因拷贝数分析：
对一个女性DMD患者及其父母进行全基因组拷贝数分析,发现患者DMD基因2-37外显子重复(即拷贝数增加),该结果与MLPA检测结果一致。患者母亲DMD基因第4内含子也存在一个重复片段,长度为10Kbp。经SNP基因分型发现患者重复的等位基因来源于母亲。
对MLPA检测未发现异常的一例女性DMD患者进行全基因组拷贝数分析,发现Xp21.1区存在31Kbp的拷贝数增加,该片段覆盖DMD基因第47外显子部分区域。第9内含子存在2kbp的拷贝数增加。
4、女性DMD患者X染色体倾斜失活状态检测：对上述两个女性DMD家系的先证者和母亲进行X染色体倾斜失活分析发现,一例先证者为随机失活,母亲为倾斜失活；另一例先证者为倾斜失活,母亲为随机失活。
5、用包装hOct4. hKlf4等因子的逆转录病毒感染羊水细胞,感染后第6天,未分化hAFDC-iPSCs克隆AP检测均呈强阳性图。将克隆样生长细胞挑到无饲养层培养基培养,可见细胞紧密排列,边界明显未分化。hAFDC-iPSCs每传10代进行核型检测,已传至40代,在传代过程中2株细胞均能维持正常核型46, XX。hAFDC-iPSCs与人胚胎干细胞系(FY-HES-1)表达含量基本相一致,Oct4、NANOG基因呈高表达,羊水细胞不表达。hAFDC-iPSCs克隆表面抗原TRA-1-60呈现强阳性；NANOG/OCT4表达阳性,显示未分化状态。
研究结论
1、DMD基因双突变是导致女性DMD患者发病的原因之一；
2、DMD基因突变合并染色体核型异常如X等臂染色体等可导致女性DMD发生；
3、利用高密度基因芯片应用于女性DMD患者发病机理的研究发现DMD基因存在拷贝数变异,部分变异可能涉及外显子。一个女性含有外显子2-37重复突变,患者的母亲DMD基因内含子存在小片段重复,而患者发生2-37重复的等位基因来源于母亲。
4、利用OCT4、KLF4两因子法诱导羊水细胞重编程,获得iPSCs,为DMD的干细胞治疗和疾病模型建立提供的良好的研究基础。
Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) is one of the most common X-linked recessive lethal genetic diseases. DMD acquired its name because it was first reported by Duchenne et al.(1868). The incidence of this disease in the general population has reached1/3,500live male births, and DMD is a commonly occurring primary muscle disease with a poor prognosis. Typical clinical features of this disease include progressive muscular atrophy and muscle weakness associated with pseudohypertrophy of the gastrocnemius muscle of the lower leg. DMD typically affects adolescent males, who generally lose the ability to stand and walk before reaching12years of age; DMD patients often die by20years of age due to heart failure or respiratory failure caused by cardiac and respiratory muscle weakness. The disease greatly impacts the healthy growth of adolescent males and has brought heavy mental and economic burdens to patients' families and to society as a whole.
To date, no effective treatments for this disease exist. Therefore, the verification of probands, the prenatal diagnosis of DMD carriers, and the elimination of the birth of affected children remain key aspects of eugenics-based genetic approaches for the prevention of DMD in China and around the world.
Before the application of genetic diagnostic techniques, the clinical diagnosis of DMD relied on typical symptoms and signs in combination with other secondary screening techniques, such as electromyography (EMG), assessments of enzyme biochemistry, and muscle biopsy. Among these screening approaches, EMG results that indicate myogenic damage and assessments of enzyme biochemistry that reveal significant increases in muscle enzyme activity represent relatively reliable bases for DMD diagnoses. However, these diagnostic approaches do not exhibit high specificity but, instead, generate false positive and false negative results. Therefore, the clinical applications of these two types of screening approaches are somewhat limited.
The DMD gene is the largest known human gene, with79exons and78introns. This gene exhibits a high mutation frequency and diverse mutant forms. Mutations in the DMD gene are the molecular genetic basis of DMD/BMD pathogenesis. Deletion mutations, duplication mutations, and point mutations are the main types of mutations of the DMD gene.
Primary methods of genetically diagnosing DMD included Southern blot hybridization, multiplex polymerase chain reaction (PCR) techniques, short tandem repeat (STR)-PCR, and reverse transcription PCR, among other approaches. Chamberlain et al. designed nine pairs of multiplex PCR primers that could detect deletions in the DMD gene in80%of DMD patients. Beggs et al. added nine additional pairs of multiplex PCR primers to the sets of primers that Chamberlain et al. had designed for DMD detection, and in combination, these18pairs of primers could detect deletions in the DMD gene in98%of DMD patients. Prior et al. first applied quantitative PCR approaches for the diagnosis of heterozygous DMD carriers; in addition, STR-PCR technologies can be used for linkage analyses of non-deletion pedigrees.
In the past, we have used various methods for the prenatal diagnosis of DMD, including gender diagnoses, multiplex PCR-based analyses of deletion mutations, and linkage analyses of STR haplotypes. These methods exhibit certain limitations and are not well adapted to the requirements for prenatal diagnoses. Gender diagnosis alone could lead to the elimination of normal male fetuses. Notably, the intragenic exchange rate for the DMD gene may be as high as11%. Thus, linkage analyses of STR haplotypes could theoretically exhibit a false-positive or false-negative rate of up to11%. Therefore, the results from these types of haplotype linkage analyses can only be regarded as probabilistic diagnoses. In addition, at times, linkage analysis cannot be performed due to lack of information regarding probands and heterozygotes or because available pedigrees are overly small. Multiplex PCR-based approaches for DMD diagnoses do not produce sufficient quantities of genetic information to span the79exons of the DMD gene; thus, these approaches can easily lead to misdiagnoses. Moreover, these approaches cannot detect duplication mutations, heterozygous carriers, or point mutations.
The objectives of our study are not only to establish a clinically feasible genetic approach for the diagnosis and prenatal diagnosis of DMD that is more efficient and less time-consuming than current diagnostic approaches for this disease but also to explore the methods and timing of early-stage prenatal diagnoses of DMD.
DMD is an X-linked recessive genetic disease; thus, in theory, DMD should predominantly affect males. However, a handful of female carriers of DMD evince clinical symptoms of this disease due to the occurrence of various unusual phenomena, such as secondary mutations. These female DMD patients are often misdiagnosed with autosomal recessive limb-girdle muscular dystrophy, which exhibits symptoms that are similar to DMD symptoms. In recent years, studies have indicated that the pathogenesis of DMD among female patients may involve X-chromosome inactivation and other types of epigenetic regulation. Therefore, the study of the molecular mechanisms underlying DMD in female patients possesses important clinical value and scientific significance.
To date, no specific treatments exist for DMD; thus, attempts to discover effective therapeutic approaches for DMD have long been a hot topic of academic research in the field of neuropathy. Induced pluripotent stem cells (iPSCs), which exhibit good immunocompatibility, are regarded as a potential source for the development of treatments of various diseases; moreover, these cells also provide an ideal model for the study of disease mechanisms. Thus, iPSCs appear to be a promising avenue to explore for discovering treatments for diseases of the nervous system. In2008, the Harvard University researchers Dimos et al. published an article in Science that detailed the ways in which they not only obtained iPSCs from the somatic cells of an82-year-old amyotrophic lateral sclerosis (ALS) patient with point mutations in the superoxide dismutase1, soluble (SOD1) gene by viral transduction, viral induction, and reprogramming but also induced the iPSCs to differentiate into motor neurons that had been damaged in the patient due to ALS. This discovery has markedly altered the difficult scenario of treating ALS patients. This finding also implies that it is possible to prepare and repair iPSCs for a patient by reprogramming the patient's somatic cells; these iPSCs can then be utilized for autologous transplantation treatments that could achieve good therapeutic effects with markedly lower levels of immune rejection than current transplantation approaches could provide. Therefore, in this study, a preliminary investigation of the establishment of iPSC lines was conducted.
Chapter1The establishment of an appropriate technology platform for the molecular diagnosis of deletion and duplication mutations of the DMD gene and the application of this platform for perinatal diagnosis
Objectives
To establish an appropriate experimental method for comprehensively screening all79exons of the DMD gene, focusing on deletion and duplication mutations; this method would be used for general and prenatal diagnoses of DMD patients and heterozygous carriers, replacing the current diagnostic approaches that only utilize probabilistic and gender-based diagnoses.
To explore the possibility of genetically detecting DMD by chorionic villus sampling, which would permit prenatal diagnoses of DMD to occur at earlier stages than current techniques would allow.
To perform whole genome amplification for a single embryonic cell and explore experimental methods for the diagnosis of DMD prior to embryo implantation.
Methods
Data sources:
1. The examined cases of this study were mainly derived from our hospital. These cases consisted of probands with typical clinical manifestations of DMD for whom examinations of serum muscle enzymes, EMG, muscle biopsy, or other assessment approaches had supported a DMD diagnosis and excluded other neuromuscular genetic diseases.
2. Amniotic fluid or chorionic villus samples were obtained from DMD carriers during pregnancy.
3. After obtaining informed consent, single embryonic cells were acquired from surplus embryonic blastomeres.
Experimental methods:
1. Proband samples were analyzed using the multiplex ligation-dependent probe amplification (MLPA) method with the P034and P035SALSA probe mixes (MRC Holland). The samples were subjected to denaturation, hybridization, and ligation reactions followed by PCR amplification and the capillary electrophoresis analysis of the resulting products, allowing for the detection of deletion and duplication mutations in the79DMD exons. This approach was compared with the multiplex quantitative PCR method.
2. A total of155pregnant women who had been verified to be heterozygous carriers of deletions or duplications in the DMD gene were examined, and the MLPA method was applied to produce prenatal diagnoses of at-risk fetuses using amniotic fluid or chorionic villus samples.
3. During the chorionic villus sampling process, maternal blood from the carriers was extracted. DNA-STR genotyping was performed for chorionic villi samples and maternal samples using the GoldeneyeTM DNA identification system to distinguish among individuals.
4. The REPLI-g Midi Kit was used to perform whole-genome amplification-multiple displacement amplification (WGA-MDA) of single embryonic cells. The WGA-MDA products were validated by the amplification of DMD-STR loci.
Results
1. Prenatal diagnoses of the fetuses of155pregnant women who carried deletions or duplications in the DMD gene detected27fetuses that were diagnosed with DMD,28fetuses that were diagnosed as carriers of mutations in the DMD gene, and100normal fetuses. The155examined fetuses included72male fetuses. The27 fetuses that had been diagnosed with DMD constituted38%of the examined male fetuses (27/72), and the45normal male fetuses that were assessed in this study constituted63%of the total number of examined male fetuses (45/72). A total of83cases of female fetuses were examined. The28fetuses that had been diagnosed as carriers of mutations in the DMD gene constituted34%of the examined female fetuses (28/83), and the55normal female fetuses that were assessed in this study accounted for66%of the total number of examined female fetuses (55/83). The pregnancies of women with fetuses that had been diagnosed with DMD were terminated, whereas the pregnancies of women with normal fetuses or fetuses that were diagnosed as carriers of DMD gene mutations were continued. Subsequent follow-up examinations were consistent with the prenatal diagnoses r%arding the DMD gene.
2. The MLPA approach could generate information for all79exons of the DMD gene, whereas the44multiplex quantitative PCR test results obtained through two experiments (with nine primer pairs in each experiment) only produced information for18exons of this gene.
3. WGA-MDA was performed for eight single embryonic cells, and the results of this analysis were verified by the amplification of DMD-STR loci. The WGA-MDA amplification yielded good results, with a quantity of amplified product that met the requirements for subsequent detection of the target gene and pre-implantation genetic screening.
Conclusions
1. The application of MLPA provides the most effective known genetic approach for the general diagnosis and prenatal diagnosis of deletion and duplication mutations in the DMD gene. In particular, this diagnostic technique is not only capable of identifying whether a DMD gene has deletion mutations or duplication mutations but can also distinguish between DMD patients and DMD carriers.
2. The MLPA method may be combined with DNA-STR genotyping technology to analyze chorionic villi samples for the early diagnosis of DMD. This combined approach can identify the presence of maternal tissue pollution in samples and thereby avoid misdiagnoses.
3. DMD gene mutations are mainly distributed between exon45and52, there are a mutation hot spots.
4. The mutation rate of DMD exons duplication have significant difference from exon deletion, which is inherited from the mother more, fewer new mutations.
5. Individual embryonic cells can be analyzed via WGA-MDA, which increases the quantity of template that is available, thereby overcoming the shortcoming that a single cell would otherwise only provide sufficient material for one detection experiment and providing experimental preparations that may be utilized to perform preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) for DMD.
Chapter2The establishment and application of molecular diagnostic techniques to detect DMD point mutations
Objectives:
Results from the first chapter of this investigation indicate that MLPA technology can detect the deletions and duplications in the DMD gene that exist in70%of DMD patients. However, in the remaining DMD patients, who constitute approximately30%of the total number of DMD patients, DMD is caused by point mutations in the DMD gene that cannot be detected by MLPA approaches. At present, because of the large size of the DMD gene, there are no medical institutions in China that can routinely detect point mutations in the DMD gene; therefore, there is no opportunity to obtain pedigrees of point mutations for use in prenatal diagnoses.
This study attempts to develop efficient sequencing methods that can detect point mutations in the79exons of the large DMD gene and thereby provide an approach that can diagnose DMD patients with point mutations who are currently unable to be genetically diagnosed with DMD through conventional clinical methods. It is hoped that these methods will ultimately increase the proportion of DMD cases that are genetically diagnosable as DMD from the current ratio of70%to90%or more. We also explore the use of the high-resolution melting (HRM) method for the preliminary screening of point mutations in the DMD gene. Furthermore, to identify single-exon deletions and point mutations, this study combines the PCR and the MLPA methods with sequencing; this combined approach can avoid misdiagnoses and provide a theoretical basis for gene repair therapies.
Methods
Data source:The examined cases of this study were mainly derived from our hospital. Probands exhibited typical clinical manifestations of DMD, and examinations of serum muscle enzymes, EMG, or muscle biopsies were utilized to support the DMD diagnosis and exclude other neuromuscular genetic diseases. The MLPA method confirmed that there were no deletion or duplication mutations in the DMD gene exons of the examined patients.
Experimental methods:
1. The Qiagen method was used to extract genomic DNA from peripheral blood samples from the examined DMD patients.
2. The MLPA approach was used to exclude patients with any deletion or duplication mutations in their DMD gene exons.
3. All79exons were sequenced in the following manner. PCR amplification was performed on the genomic DNA samples from the study subjects. Subsequently, the PCR products were purified, sequencing reactions were performed, and the sequencing PCR products were purified. An ABI3100Genetic Analyzer was then used to assess these purified sequencing products through capillary electrophoresis, and the results from this process were analyzed and processed with the GeneMapper ID v3.1and Mutation Surveyor software packages to obtain sequencing results.
4. A LightScanner HRM system was used to detect known point mutations and certain unknown point mutations in the examined samples.
5. MLPA results indicating single-exon deletions in patients were subjected to verification through PCR and DNA sequencing.
Results
1. We performed genetic sequencing of blood samples from16probands and found that in13of these patients, a single nucleotide substitution had occurred in the DMD gene. Among the remaining three probands who were examined, one proband had a DMD gene that was missing a single nucleotide, one proband had a DMD gene that was missing two nucleotides, and the final proband had a DMD gene with an insertion of5nucleotides. These point mutations resulted in premature termination codon mutations, frameshift mutations, and other deleterious alterations of the DMD gene.
2. In total,37cases that the MLPA method had diagnosed as single-exon deletions from the DMD gene were examined, and in30of these cases,81.08%of the examined cases, an exon deletion in the DMD gene was detected through PCR and sequencing approaches. In the remaining seven cases, which accounted for18.92%of the examined instances of alleged single-exon deletion, the PCR method did not identify deletions, and sequencing provided conclusive evidence that exon point mutations were present in these seven cases.
3. HRM analyses that were performed for DNA samples from ten cases revealed peaks to indicate point mutations for exons of100-300bp in size; however, in contrast to expectations, these peaks failed to appear for exons of other sizes.
Conclusions
1. The genomic sequencing method is a reliable detection method for point mutations in the DMD gene. After the MLPA method has been utilized to exclude DMD patients with deletion or duplication mutations in this gene, the sequencing of the complete set of DMD exons could be utilized to genetically diagnose DMD in the remaining patients, who have DMD point mutations. It can be applied to prenatal diagnosis.
2. A determination of a single-exon deletion by the MLPA approach must be combined with the application of PCR and sequencing methods, which can identify whether the MLPA results reflect a true deletion or a point mutation and thereby improve upon the accuracy of the MLPA approach for the detection of single-exon deletions in the DMD gene.
3. High-resolution melting curves can be used to screen for point mutations. However, under the conditions that were employed in this study, this method is only effective to detect point mutations in exons of100-300bp in size. Thus, further exploration is required to improve methods for screening for point mutations.
Chapter3The study of DMD pathogenesis in females and the establishment of iPSC lines from amniotic fluid
Objectives
DMD/BMD is an X-linked recessive genetic disease; thus, in theory, this disease should predominantly affect males. Clinical symptoms of this disease in females appear in a handful of carriers due to various phenomena, such as secondary mutations. Therefore, female patients with this disease are often misdiagnosed with autosomal recessive limb-girdle muscular dystrophy, which exhibits symptoms that are similar to the manifestations of DMD/BMD. In recent years, studies have shown that the pathogenesis of female patients might involve epigenetic mechanisms, such as X-chromosome inactivation. Therefore, the study of the molecular mechanisms of DMD pathogenesis in female patients has important clinical value and scientific significance. In this study, to detect the presence of small deletions/duplications in the genomes of female DMD patients, high-resolution Affymetrix CytoScan HD arrays are utilized to perform genome-wide analyses of these patients. Methylation-specific PCR is employed to detect X-chromosome inactivation and examine the correlation of this inactivation to the pathogenesis of DMD in female patients.
There are currently no specific treatments for DMD; instead, only symptomatic treatments and supportive care exist for this disease. The search for effective treatments for DMD has long been a hot topic of academic research in the field of neuropathy. iPSCs, which exhibit good immunocompatibility, are regarded as a potential source for the development of treatments of various diseases; moreover, these cells also provide an ideal model for the study of disease mechanisms. Many cell components exist in amniotic fluid; in fact, at present, the amniotic fluid is thought to include a heterogeneous population of cells that have been derived from fetuses and amniotic membranes. Amniotic fluid cells, which consist of a variety of cells from different sources, exhibit excellent pluripotency potential and can be induced to differentiate into cells of all embryonic germ layers. This study seeks to use the two factors of octamer-binding transcription factor4(Oct4) and Kruppel-like factor4(Klf4) to induce the re-programming of amniotic fluid cells and thereby obtain iPSCs; this process would provide a suitable research foundation for developing stem cell therapies for DMD and establishing models of this disease.
Methods
Data sources:
1. Data were obtained from the four female DMD patients who have been treated in our hospital since2006.
2. Amniotic fluid cell samples were collected from a woman who had undergone a medically recommended amniocentesis when she was20weeks pregnant with twins. After prenatal diagnostic procedures using the sampled amniotic fluid from this woman had been completed, the remaining amniotic fluid was employed for this study after obtaining informed consent from the donor couple and approval from the ethics committee of the Third Affiliated Hospital of Guangzhou Medical College.
Experimental methods:
1. The Qiagen method was used to extract genomic DNA from the peripheral blood samples of female DMD patients. Analytical procedures and quality control were performed in accordance with the operating protocols for the Affymetrix CytoScan HD array.
2. To detect the status of X-chromosome inactivation in the examined patients, genomic DNA samples from these patients were treated with sulfates and then amplified with methylation-specific primers. The amplification products were analyzed via capillary electrophoresis.
3. The iPSCs were prepared as follows. First, the examined amniotic fluid cells were collected, passaged, and infected. A primary culture of human amniotic fluid-derived cells (hAFDC)-iPSCs was created and passaged. These cells were then subjected to reverse transcription (RT)-PCR detection of the expression of pluripotency genes, the detection of alkaline phosphatase (AP), immunofluorescence staining, the detection of in vitro differentiation capacity, the detection of in vivo differentiation potential, and karyotype analysis.
Results
1. Genome-wide copy number analyses were performed for one female DMD patient and her parents. These analyses revealed a DMD gene in this patient with the duplication of exons2-37(i.e., an abnormally high copy number); this result was consistent with findings from the MLPA approach. The patient's mother possessed a DMD gene with a duplicated10kbp fragment in intron4. Single nucleotide polymorphism (SNP) genotyping suggested that the patient had obtained her allele containing the aforementioned-duplication mutation in DMD from the mother.
2. Genome-wide copy number analysis was performed to assess one female DMD patient for whom MLPA approaches had failed to find abnormalities. This analysis revealed an abnormally high copy number for the examined patient for a31kbp fragment in the Xp21.1region that overlapped with part of exon47of the DMD gene. In addition, this patient exhibited an abnormally high copy number for a2kbp fragment in intron9of the DMD gene.
3. Retroviruses containing the hOct4and hKlf4genes were used to infect the amniotic fluid cells. At six days after this infection, undifferentiated hAFDC-iPSC clones exhibited strong positive signals in AP detection analyses. Cells with clone-like growth were picked and cultured in feeder-free medium. The undifferentiated cells formed densely packed cell masses with clear borders. The karyotypes of the cells were examined every ten passages to assess the hAFDC-iPSCs. These cells have undergone40passages. During the passaging process, the two cell lines were both able to maintain a normal karyotype of46, XX. hAFDC-iPSCs and the human embryonic stem cell line FY-hES-1exhibited rather similar expression levels of pluripotency genes; in particular, the Oct4and Nanog genes were highly expressed in both of these cell types, whereas neither of these genes was expressed in differentiated amniotic fluid cells. The hAFDC-iPSCs clones were strongly positive for the transformer (TRA)-1-60surface antigen and positive for NANOG/OCT4expression, indicating that these clones existed in an undifferentiated state.
Conclusions
1. Double mutations is one of the reasons for the female DMD patient.
2. DMD gene mutation combined chromosomal abnormalities is another reasons.
3. High-density gene arrays were utilized to investigate the pathogenesis of female DMD patients. These analyses determined that one female DMD patient had an abnormal copy number for portions of the DMD gene and that this phenomenon might involve exons of this gene. A female DMD patient with a duplication of exons2-37of the DMD gene appears to have inherited this abnormal allele from her mother. One of her mother's DMD genes contains small intronic fragment repeats, suggesting that this DMD gene might be unstable. Certain female DMD patients exhibit secondary mutations of their DMD genes.
2. The two factors of OCT4and KLF4were utilized to induce the reprogramming of amniotic fluid cells to obtain iPSCs, thus establishing a suitable foundation for the development of DMD stem cell therapies and for the establishment of models of DMD.
引文
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