杜氏肌营养不良症产前基因诊断后十年追踪

目的：了解产前期诊断后出生的女孩健康情况。方法：应用限制性内切酶片段长度多态性（RFLP），分析血清CK及健康体检随访。结果：产前基因诊断女孩携有87-15的9.4kb片段，CK265U/L是DMD致病式因携带，观生长发育正常，排除她不是Duchenne型肌营养不良症（DMD）患或有症状的DMD的女性携带。讨论：本病是由DMD致病基因遗传所致，两名男性患，发病年龄均在2-3岁，十年后随访，Ⅱ4死于本病，Ⅲ1不能行走，生活不能自理。产前诊断女孩（Ⅲ2）只有血清酶学具有DMD阳性特征，随访支持产前诊断结果，对指导女性DMD基因携带的生育具有重要意义。     

Duchenne型肌营养不良的康复评估及治疗研究进展

Duchenne型肌营养不良（DMD）又称假肥大型进行性肌营养不良,是儿童最常见的X连锁隐性遗传性肌肉病,表现为对称性、进行性肌无力,运动功能的丧失是其核心症状。本病目前尚无有效治愈的方法,康复治疗对改善患儿生活质量、延缓病情进展至关重要。本文对近年来DMD的康复评估及治疗研究进展作一综述。     

有Duchenne肌营养不良家族史孕妇的产前基因诊断

目的 对有Duchenne肌营养不良(Duchenne muscular dystrophy,DMD)家族史的孕妇进行产前诊断研究.方法 PCR检测胎儿的性别决定基因(SRY)和DMD基因常见缺失的18个外显子,同时用DMD基因的6个(CA)n重复序列STR位点进行连锁分析,对31例有DMD家族史的孕妇进行产前诊断;随...     

血清VEGF的表达与肌营养不良症的关系

目的检测肌营养不良症患者的血清血管生长因子（VEGF）水平,鉴定其是否与肌营养不良症的疾病发展有关。方法对46例肌营养不良患者,其中32例Duchenne肌营养不良症（DMD）、9例Becker肌营养不良症（BMD）、5例强直性肌营养不良症（DM）患者的血清VEGF水平进行检测。15例健康人和8例疾病患者为对照组。结果DMD患者血清VEGF为（274．7±2．52）pg／ml,BMD患者的为（358．8±9．64）pg／ml,DM患者为（165．0±6．34）pg／ml,而健康对照组为（148．3±2．91）pg/ml,疾病对照组为（153．7±5．42）pg／ml。与DM组和对照组相比,BMD的VEGF水平显著提高。而DMD组中卧床的患者相对于坐轮椅的DMD、DM组和对照组则VEGF水平显著升高。结论VEGF可以反映肌肉组织低氧和／或缺血状况,并且与DMD和BMD患者的疾病发展过程有关。     

临床护理观察路径在造血干细胞移植治疗假肥大型肌营养不良中的应用

假肥大型肌营养不良又称杜兴（氏）肌营养不良（Duchenne’8musculardystrophy,DMD）,是一种X连锁隐性遗传性肌病,以进行性四肢近端骨骼肌萎缩无力、小腿腓肠肌假性肥大为特征,同时累及心肌和呼吸肌,目前尚无有效的治疗方法^[1]。近年来学者们对干细胞治疗DMD做了许多基础和l临床研究,为DMD患儿提供了一个崭新的治疗路径^[2]。     

中国西北地区102例儿童Duchenne型肌营养不良基因突变及家系分析研究

目的　了解西北地区Duchenne型肌营养不良症(DMD)患者基因突变特点。方法　收集2014年7月～2020年6月的102例DMD患者作为研究对象,采用多重连接依赖探针扩增（multiplex ligationdependent probe amplification, MLPA）技术、二代基因测序（next-generation sepuencing, NGS)技术及Sanger测序方法进行基因检测,分析西北地区DMD患者基因缺失突变、重复突变、点突变的分布区域特点,且对2个DMD家系基因特点进行分析。结果　102例DMD患者,72.5%来源于遗传,27.5%为新发突变。基因突变类型中,85.3%为片段缺失,6.9%为片段重复,7.8%为点突变。片段缺失突变中,≤5个数目的外显子缺失最为常见,占比为66.6%;外显子44~54是最常见的缺失区域,外显子2是最常见的重复区域。2个家系研究,家系1中突变位点为缺失突变的热点区域,外显子45~51区域;家系2中主要是重复突变,集中在外显子2和17~18区域。结论　大样本对西北地区DMD患者及家系的基因分析,可以进一步了解DMD患者基因谱,为基因治疗建立基础。     

Duchenne型肌营养不良症的治疗进展

Duchenne型肌营养不良症(DMD)是一种X连锁隐性遗传的致死性肌病,一般6岁左右发病,9~14岁渐至不能行走,多于20岁左右死于心、肺功能衰竭。目前DMD的治疗方法匮乏,本文对DMD治疗进展进行综述。     

Duchenne型肌营养不良症病人中出现"返祖纤维"的免疫荧光和分子生物学研究

目的：探讨临床诊断为Duchenne型肌营养不良（Duchenne's muscular dystrophy,DMD)病人的免疫荧光检测中出现“返祖纤维”现象的可能机制。方法；对病人的肌肉组织行免疫荧光检查抗肌营养不良蛋白（dystrophin)抗体，并同时对病人DMD基因中的25个外显子进行检测。结果：21例病人中有5例出现“返祖纤维”现象，即在免疫荧光检测中有单个或一小簇肌纤维呈阳性反应，其余呈阴性表现，多重PCR检测该5例病人，其中3例有外显子缺失。结论：“返祖纤维”现象提示：病人的个别肌纤维可能存在完整的抗肌营养不良蛋白基因表达，其发病机制可能有别于传统意义上的DMD发病机制。“返祖纤维”现象可用来评估病人的预后，并为临床治疗提供一些新的思路。     

贝氏肌营养不良症与杜氏肌营养不良症患者骨骼肌脂肪浸润程度的对比研究

目的分析贝氏肌营养不良症（BMD）患者大腿骨骼肌在磁共振成像（MRI）上所显示的骨骼肌脂肪浸润规律,探讨其与杜氏肌营养不良症（DMD）患者之间的差异,为针对性康复治疗提供指导依据。 方法纳入23例BMD患者和47例DMD患者,所有患者均未经糖皮质激素治疗,均行双侧大腿骨骼肌MRI检查,并通过T1加权成像和应用改良的Mercuri分级评分法,对骨骼肌脂肪浸润程度进行0～5分的6级评分。采用描述性统计分析BMD的骨骼肌脂肪浸润进展程度,采用秩合检验分析BMD和DMD的组间差异。 结果BMD患者大收肌的重度脂肪浸润所占百分比最高,而后受累的依次为股二头肌、股四头肌、半膜肌和半腱肌,缝匠肌、股薄肌和长收肌的重度脂肪浸润所占百分比最低。BMD患者的大收肌、股二头肌和股四头肌在8～9岁可见中到重度脂肪浸润,半膜肌和半腱肌在10～11岁可见中到重度脂肪浸润,缝匠肌、股薄肌和长收肌在15岁以后可见轻到中度脂肪浸润。BMD组骨骼肌脂肪浸润程度评分总和的中位数在8、9、10和11岁依次为10、22、28和25分,DMD组依次为29、34、34和30分。BMD与DMD相比,8岁患者的脂肪浸润程度评分在大收肌（P=0.017）、股二头肌（P=0.013）、股外侧肌（P=0.021）、股直肌（P=0.007）、股内侧肌（P=0.008）、股中间肌（P=0.009）及评分总和（P=0.011）之间的差异均有统计学意义;9岁患者的脂肪浸润程度评分在大收肌（P=0.007）、股直肌（P=0.013）、股内侧肌（P=0.028）、股中间肌（P=0.028）及评分总和（P=0.020）之间的差异具有统计学意义。 结论BMD患者的大腿骨骼肌脂肪浸润存在一个特定受累模式,相同年龄的BMD与DMD患者骨骼肌脂肪浸润程度存在差异,可以协助指导BMD及DMD患者的康复治疗。     

Duchenne型肌营养不良症的治疗

Duchenne型肌营养不良症(Duchenne muscular dystrophy, DMD)是一种X连锁致死性遗传性肌病,由Dystrophin基因突变导致抗肌萎缩蛋白缺失所致。DMD尚缺乏有效的治疗方法,但随着对该病发病机制和病理变化过程的认识不断深入,其治疗选择越来越多。这些治疗方法旨在恢复Dystrophin蛋白表达或弥补Dystrophin蛋白缺失。     

Duchenne muscular dystrophy in a female patient with a karyotype of 46,X,i(X)(q10)

Duchenne muscular dystrophy (DMD) is a severe recessive X-linked form of muscular dystrophy caused by mutations in the dystrophin gene and it affects males predominantly. Here we report a 4-year-old girl with DMD from a healthy family, in which her parents and sister have no DMD genotype. A PCR-based method of multiple ligation-dependent probe amplification (MLPA) analysis showed the deletion of exons 46 and 47 in the dystrophin gene, which led to loss of dystrophin function. No obvious phenotype of Turner syndrome was observed in this patient and cytogenetic analysis revealed that her karyotype is 46,X,i(X)(q10). In conclusion, we describe the first female patient with DMD who carries a de novo mutation of the dystrophin gene in one chromosome and isochromosome Xq, i(Xq), in another chromosome.     

中国北方59个假肥大性肌营养不良家系中抗肌萎缩蛋白基因突变分析

目的 分析研究我国北方地区假肥大性肌营养不良[分为杜克肌营养不良(DMD)和贝克肌营养不良(BMD)]患者抗肌萎缩蛋白基因突变的类型,断裂点的分布,并为临床应用奠定基础.方法 采用多重连接探针扩增法(MLPA)检测59个来自北方地区DMD(51例)及BMD(8例)家系的男性患者及其父母的抗肌萎缩蛋白基因突变情况.结果 从59个DMD/BMD家系的59例DMD/BMD患者中,检测到33例外显子缺失,6例外显子重复和l例点突变;内含子44是最常见的断裂点(n=13,33.3%),内含子50和45为第二位(n=11,28.2%)和第三位(n=8,20.5%).并发现2例为Leiden数据库中未报道的新发突变,即D,149的2个位点的重复突变(外显子3～7和外显子44)和D165的点突变[5208del(A)].1例患者为中国人群中未见报道的外显子22缺失.19例患者用MLPA方法未检测到突变.结论 缺失突变主要发生在外显子45～50的中央热区,重复突变主要发生在基因的5'端,内含子44是中国北方人群DMD基因缺失最常见的断裂点.     

Dystrophin expression in muscle following gene transfer with a fully deleted ("gutted") adenovirus is markedly improved by trans-acting adenoviral gene products

Helper-dependent adenoviruses (HDAd) are Ad vectors lacking all or most viral genes. They hold great promise for gene therapy of diseases such as Duchenne muscular dystrophy (DMD), because they are less immunogenic than E1/E3-deleted Ad (first-generation Ad or FGAd) and can carry the full-length (Fl) dystrophin (dys) cDNA (12 kb). We have compared the transgene expression of a HDAd (HDAdCMVDysFl) and a FGAd (FGAdCMV-dys) in cell culture (HeLa, C2C12 myotubes) and in the muscle of mdx mice (the mouse model for DMD). Both vectors encoded dystrophin regulated by the same cytomegalovirus (CMV) promoter. We demonstrate that the amount of dystrophin expressed was significantly higher after gene transfer with FGAdCMV-dys compared to HDAdCMVDysFl both in vitro and in vivo. However, gene transfer with HDAdCMVDysFl in the presence of a FGAd resulted in a significant increase of dystrophin expression indicating that gene products synthesized by the FGAd increase, in trans, the amount of dystrophin produced. This enhancement occurred in cell culture and after gene transfer in the muscle of mdx mice and dystrophic golden retriever (GRMD) dogs, another animal model for DMD. The E4 region of Ad is required for the enhancement, because no increase of dystrophin expression from HDAdCMVDysFl was observed in the presence of an E1/E4-deleted Ad in vitro and in vivo. The characterization of these enhancing gene products followed by their inclusion into an HDAd may be required to produce sufficient dystrophin to mitigate the pathology of DMD by HDAd-mediated gene transfer.     

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.     

Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse - an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.     

CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.     

Restoration of dystrophin expression in mdx mice by intravascular injection of naked DNA containing full-length dystrophin cDNA

Duchenne muscular dystrophy (DMD) is a lethal, X-linked, recessive disease caused by a defect in the dystrophin gene. No effective therapy is available. Dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. However, successful treatment for DMD requires restoration of dystrophin in the affected muscle fibers to at least 20% of the normal level. Current gene transfer methods such as intramuscular injection of viral vector or naked DNA can only transfect a small area of muscle, and therefore is of little clinical utility. We have developed a semisystemic method for gene transfer into skeletal muscle of mdx mice, an animal model for DMD. Naked DNA was injected through the tail artery or vein of mice, in which the aorta and the vena cava were clamped at the location just below the kidneys. The DNA solution was thus forced into the blood vessels of both legs. Luciferase gene expression was detected in all muscle groups in both legs. The effects of injection speed, injection volume, and ischemia time on gene expression were also optimized. LacZ staining was used to check the spread of gene expression in muscle. Although the percentage of transfected fibers was modest (approximately 10%), beta-galactosidase was found in all muscle groups of both legs. Finally, plasmid DNA encoding full-length dystrophin gene was injected into mdx mice and widespread restoration of dystrophin protein was observed in all muscles of both hind limbs. In conclusion, these results demonstrate that the semisystemic delivery of naked DNA is a potential approach towards the long-term goal of gene therapy for DMD.     

Chimeric RNA/ethylene-bridged nucleic acids promote dystrophin expression in myocytes of duchenne muscular dystrophy by inducing skipping of the nonsense mutation-encoding exon

Editing of dystrophin mRNA by induction of exon skipping, using antisense oligonucleotides, has been proposed as one way to generate dystrophin expression in Duchenne muscular dystrophy (DMD) patients. Here, antisense chimeric oligonucleotides consisting of RNA and a new modified nucleic acid are tested for activity to induce skipping of an exon containing a nonsense mutation. In a Japanese DMD case, a nonsense mutation (R1967X) due to a single nucleotide change in exon 41 of the dystrophin gene (C5899T) was identified. Oligonucleotides consisting of 2'-O-methyl RNA and a new 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) were designed to bind the mutation site of exon 41, and their ability to induce exon 41 skipping in dystrophin mRNA was evaluated. Finally, among the specific oligonucleotides tested, an 18-mer RNA/ENA chimera was found to have the strongest activity, inducing exon 41 skipping in nearly 90% of dystrophin mRNA. Accordingly, nearly 90% of cultured myocytes were shown to be dystrophin positive by immunohistochemical analysis. Western blot analysis disclosed the presence of nearly normal-sized dystrophin up to 1 week after the transfection. Our results suggest that an RNA/ENA chimera can be used to express dystrophin in DMD.     

Long-term expression of full-length human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins

One of the possible therapies for Duchenne muscular dystrophy (DMD) is the introduction of a functional copy of the dystrophin gene into the patient. For this approach to be effective, therapeutic levels and long-term expression of the protein need to be achieved. However, immune responses to the newly expressed dystrophin have been predicted, particularly in DMD patients who express no dystrophin or only very truncated versions. In a previous study, we demonstrated a strong humoral and cytotoxic immune response to human dystrophin in the mdx mouse. However, the mdx mouse was tolerant to murine dystrophin, possibly due to the endogenous expression of dystrophin in revertant fibres or the other nonmuscle dystrophin isoforms. In the present study, we delivered human and murine dystrophin plasmids by electrotransfer after hyaluronidase pretreatment to increase gene transfer efficiencies. Tolerance to murine dystrophin was still seen with this improved gene delivery. Tolerance to exogenous recombinant full-length human dystrophin was seen in mdx transgenic lines expressing internally deleted versions of human dystrophin. These results suggest that the presence of revertant fibres may prevent the development of serious immune responses in patients undergoing dystrophin gene therapy.