Dystrophinopathy分子生物学及临床研究

  本文关键词：Dystrophinopathy分子生物学及临床研究，由笔耕文化传播整理发布。

        DMD基因变异导致的dystrophin蛋白缺陷引起的一组遗传性疾病称为dystrophinopathy。DMD基因定位在Xp21.2，编码蛋白dystrophin。男女均可发病，但男性患者占绝大多数，临床表型包括：杜兴型肌营养不良（Duchenne muscular dystrophy, DMD）、贝克型肌营养不良（Beckermuscular dystrophy, BMD）（包括无症状高肌酸激酶血症、肌痉挛/肌痛、股四头肌肌病）、X连锁扩张型心肌病（X-linked dilated cardiomyopathy,XLDCM）、有/无症状DMD基因变异携带者。其中DMD是发病率最高、临床表现最重类型，多于5-6岁起病，表现走路慢，易摔倒，走路姿势异常，进展加重出现明显双下肢、腰带肌无力，蹲下站起、上下楼梯费力，约10-12岁左右不能行走，20-30岁死于心肺功能衰竭。BMD临床症状出现较DMD晚，可保持行走能力到16岁，病情进展相对缓慢，寿命较长。女性DMD基因缺陷携带者和邻近基因缺失女性患者临床表现差异巨大，重者可为典型DMD表现，轻者表现为轻度近端肌无力、腓肠肌假性肥大，甚至肌肉功能基本正常。Dystrophinopathy致病基因DMD至今仍是已发现的最大的基因，包含79个外显子，编码蛋白dystrophin，dystrophin蛋白包括4个不同的区域，氨基端与肌动蛋白（actin）或其相关蛋白相连，棒状端拥有长的24个膜收缩蛋白样（spectrin-like）重复区和位于两端的4个绞链区，胱氨酸富集区和羧基段选择性剪接区。Dystrophin蛋白连接细胞骨架与基底膜，并与其他蛋白（dystroglycan、sarcoglycan）形成肌营养不良糖蛋白复合体（dystrophinglycoprotein complex, DGC）维持细胞膜稳定性。基因变异导致整个蛋白复合体破坏使肌细胞膜脆性增加、稳定性破坏，强烈的肌肉收缩加剧肌细胞膜破坏使钙离子内流加速肌纤维的破坏。本研究从骨骼肌遗传资源标本库中筛选：1）部分dystrophinopathy患者进行分子病理和基因测序，重点选择2例有症状女性DMD基因变异携带者家系进行临床和基因变异分析；2）确诊的42例dystrophinopathy患者进行下肢骨骼肌MRI研究分析；3）DMD患者激素治疗分析，旨在对dystrophinopathy进行临床、影像、基因和激素治疗系列研究，发现临床、病理及分子生物学特点及治疗观察经验。第一部分Dystrophinopathy分子生物学研究目的：Dystrophinopathy是dystrophin蛋白结构、功能异常相关的一组遗传性骨骼肌疾病，致病基因DMD定位在Xp21.2，包含79个外显子，编码蛋白dystrophin。临床表型包括：Duchenne muscular dystroph（yDMD）、Becker muscular dystrophy（BMD）、X连锁扩张型心肌病（X-linked dilatedcardiomyopathy，XLDCM）、有/无症状DMD基因变异携带者。约8-22%的女性DMD基因变异携带者出现程度不同的肌无力表现，称为有症状携带者（manifesting carriers, MCs）。目前女性MCs发病机制尚未完全明确，倾斜的X染色体失活模式是目前研究热点。因病例较少尚未发现女性MCs基因热点突变，基因变异-X染色体失活-临床表现关系不明。本文对dystrophinopathy患者和2例女性MCs家系进行临床、骨骼肌病理和基因变异分析，旨在发现dystrophinopathy基因变异特点，重点探讨女性MCs的临床、基因变异特点及其可能的发病机制。方法：1临床病例入选标准：1）临床表现：缓慢进行性加重四肢近端肌、腰带肌无力、萎缩，腓肠肌假性肥大，Gower征（+）；2）高CK血症，肌电图呈肌源性损害；3）活检骨骼肌组织化学染色呈肌营养不良改变，抗-dystrophin-N，-C，-R单克隆抗体免疫组织化学染色dystrophin蛋白完全/部分缺失。2活检骨骼肌病理分析：入选病例行骨骼肌活检组织化学染色和抗dystrophin-N、-C、R单克隆抗体免疫组织化学染色病理分析。3DMD基因测序：多重PCR-DHPLC检测外显子缺失和重复突变，直接测序检测点突变。4女性MCs2例及家系临床和基因分析：4.1家系资料：选取有肢体近端肌无力、萎缩的2例女性患者，并且家族中有男性DMD/BMD患者的家系。4.2对家系患者及部分亲属行心电图、心肌核素显像及下肢肌肉MRI检查，评价心肌、骨骼肌受累情况。4.3骨骼肌活检组化和免疫组化染色：家系1先证者及家系2先证者及Ⅳ-4行开放式左肱二头肌活检病理分析（方法同上）。4.4核型分析：女性MCs和女性无症状携带者做常规GRQ带显色核型分析。4.5DMD基因测序：方法同上。4.6X染色体失活（X chromosome inactivation，XCI）分析：利用患者外周血DNA的扩增雄激素受体（androgen receptor，AR）区域，根据等位基因AR区域CpG数量不同，可区分父母X染色体。HpaII限制性内切酶消化作用确定其父母双方的X染色体的活性。结果：1临床资料：80例dystrophinopathy患者中，DMD患者67例，BMD患者10例，女性MCs2例，无症状高CK血症1例。2骨骼肌活检组化和免疫组化病理：全部病例活检骨骼肌组织化学染色呈肌营养不良改变，轻重程度与病程相关；抗dystrophin-N、-C、-R单克隆抗体免疫组织化学染色肌细胞膜dystrophin蛋白完全（67例）/部分缺失（13例）。3基因测序：14例dystrophinopathy患者行DMD基因测序分析：1例BMD未发现致病基因突变，1例DMD患者发现点突变c.4178T>A，余12例患者发现DMD外显子缺失突变。4女性MCs2例及家系临床和基因研究4.1临床表现：2个家系均呈X-连锁隐性遗传，先证者均表现不同程度肢体近端肌无力，无明显腓肠肌肥大。家系1先证者发病晚，临床症状较轻，家系2先证者发病早症状较重。4.2家系患者心肌受累情况：家系1先证者心电图和心肌核素显像未见异常。家系2先证者心肌核素显像：心尖处血流灌注轻度降低，左室射血分数：70%，左室结构和功能大致正常。4.3下肢肌肉MRI：家系1先证者：下肢肌肉受损不对称，肌群受损与DMD相似。家系2先证者：仅股薄肌、伸踇长肌、屈趾长肌相对保留，剩余肌肉均被脂肪和结缔组织替代。家系2Ⅳ-4肌群受损与BMD相似。4.4骨骼肌活检组化和免疫组化病理：2个家系先证者和家系2Ⅳ-4活检骨骼肌病理均为肌营养不良改变，抗dystrophin-N、-C、-R单克隆抗体免疫组织化学染色，肌纤维膜dystrophin蛋白在部分肌细胞膜上缺失/减弱，，家系1先证者肌纤维膜dystrophin蛋白部分缺失呈马赛克样分布；家系2两名患者dystrophin-N、-R端完全缺失，-C表达减弱。4.5核型分析：家系1先证者和家系2先证者和Ⅱ-5、Ⅲ-7常规GRQ带染色体核型分析为正常女性核型。4.6DMD基因测序：家系1先证者和Ⅱ-7发现外显子61杂合性缺失。家系2先证者、Ⅱ-5和Ⅲ-7发现外显子12-43杂合性缺失，家系2Ⅳ-4发现外显子12-43纯合性缺失。2例先证者未发现有临床意义点突变。4.7XCI分析：家系1先证者、家系2先证者和Ⅲ-7X染色体失活检测均不能区分父母的等位基因(uninformative)。结论：1活检骨骼肌组织化学、抗-dystrophin单克隆抗体免疫组织化学染色病理分析有助于临床诊断dystrophinopathy，指导目的基因分析，判断临床表型和疾病预后。2对于临床诊断dystrophinopathy，基因分析首选片段缺失/重复筛查，再行点突变检测。3女性MCs临床症状严重程度与发病年龄有关，与骨骼肌病理像轻重程度相对应，与dystrophin蛋白表达无关。4女性MCs2例及家系均DMD基因外显子片段缺失，均未检测到具有临床意义点突变，女性MCs应常规进行核型和XCI分析；女性MCs临床表型-基因变异不符合阅读框学说，男性患者基因变异-临床表型多数符合阅读框学说。第二部分Dystrophinopathy下肢骨骼肌MRI研究目的：Dystrophinopathy主要发病机制为肌细胞骨架蛋白dystrophin缺失、肌细胞稳定性破坏，持续肌细胞变性、坏死和再生，结缔组织增生，随病程进展，单位容积肌细胞数目减少，脂肪组织替代。研究发现DMD患者肌肉损害T1加权成像表现为肌肉组织中出现短T1信号，压脂呈低信号，说明肌肉组织间脂肪组织浸润，随病情进展脂肪浸润程度增加，并表现出特异性选择性肌肉损害：大腿股薄肌、缝匠肌及半膜肌相对保存，股四头肌、比目鱼肌、腓肠肌损害明显，其中腓骨肌受损是DMD肌肉损害的特征之一。分析2004-2012年我科确诊的42例dystrophinopathy患者临床、下肢骨骼肌MRI特点，旨在总结、分析选择性肌群受累特点，探索分子影像学水平dystrophinopathy的诊断要点，及其与其它类型PMD的鉴别诊断要点。方法：1研究对象入组标准：1）儿童、青少年起病，下肢近端肌无力、肌萎缩，腓肠肌假性肥大，Gower征（+）；2）血CK显著升高，肌电图呈肌源性损害；3）肱二头肌活检组织化学染色病理分析大量的肌纤维变性、坏死和再生，结缔组织增生；抗dystrophin-N，-C，-R单克隆抗体免疫组化染色，肌细胞膜dystrophin蛋白完全缺失诊断DMD，部分缺失诊断BMD。2归纳分析42例入组患者临床资料，包括：性别、发病年龄、血肌酸激酶值，半定量评估、统计学分析下肢近/远端肌力，大小腿骨骼肌MRI受累肌群。3总结分析dystrophinopathy下肢骨骼肌MRI受累肌群特点及与临床表现相互关系。结果：1临床资料：下肢肌力与年龄呈负相关(大腿：r=-0.550, P=0.001<0.05,小腿：r=-0.400, P=0.021<0.05），近端肌力与远端肌力呈正相关（r=0.642,P<0.01）。2选择性肌群受累程度分析：DMD组下肢骨骼肌MRI，大腿肌肉受累程度重于小腿（P<0.001）；大腿肌群损害出现早且重于小腿。大腿肌群中股薄肌受累程度最轻，半腱肌次之，前群肌中股外侧肌最重；小腿肌群中胫骨前肌受累程度最小，后群肌中腓肠肌外侧头受累重于比目鱼肌和腓肠肌内侧头，前群肌中腓骨长肌受累最重。6例BMD肌群受累特点与DMD相似，1例无症状高CK血症和1例DMD基因缺陷携带者仅在股四头肌和腓肠肌呈轻度受累。3肌肉受累程度与临床资料对比分析：血CK与下肢肌肉受损程度无相关性（P=0.266>0.05）。大腿肌群与小腿肌群损伤程度均与年龄存在正相关性（大腿： r=0.720，P=0.009<0.05；小腿： r=0.680，P <0.01）；下肢近端肌力与大腿肌肉损伤程度存在负相关性（r=-0.611, P <0.01），远端肌力与小腿肌肉损伤程度存在负相关性（r=-0.462，P=0.012<0.05）。结论：1Dystrophinopathy患者下肢骨骼肌MRI存在特征性选择性肌群受累，受累特点与临床表现相对应，甚至在亚临床期MRI即可呈现肌肉损害特点。2Dystrophinopathy下肢骨骼肌MRI选择性肌群受累具有一定的规律性，此特点有助于分子影像水平诊断dystrophinopathy，鉴别诊断其他类型进行性肌营养不良，提示临床分型、指导致病基因测序。3下肢骨骼肌MRI可以客观评价dystrophinopathy患者病情，根据受累肌群特点指导康复治疗，评估药物治疗疗效。第三部分杜兴型肌营养不良糖皮质激素治疗分析目的：DMD是进行性肌营养不良中发病率最高的类型。约10-12岁左右不能行走，20-30岁死于心肺功能衰竭。血肌酸激酶（creatine kinase，CK）显著增高数十至数百倍，肌电图呈肌源性损害。目前DMD无有效的根治方法，主要以保存患者运动功能、防治并发症为治疗目的，包括：糖皮质激素治疗、适当康复锻炼及外科矫形等。其中糖皮质激素治疗是国内外公认的可延缓患者肌力丧失速度，保存运动功能，提高生活质量的药物。回顾性分析96例DMD患者临床、实验室表现，评判糖皮质激素治疗效果。方法：1研究对象入组标准：诊断基于：明显的下肢为主的肌无力、肌萎缩，血CK显著升高，肌电图呈肌源性损害，肱二头肌活检组织化学染色大量的肌纤维变性、坏死和再生，结缔组织增生，抗dystrophin-N，-C，-R单克隆抗体免疫组化染色，肌细胞膜dystrophin蛋白完全缺失。2分组及激素治疗方法：根据首诊年龄进行分组，再根据体重、年龄及血CK水平给予地塞米松5-10mg静脉输液治疗10-15天复查血CK，继续给予口服醋酸泼尼松0.5-0.75mg/kg.d维持治疗，1月后复查血CK，继续醋酸泼尼松维持治疗。3统计学分析激素治疗前后患者血CK、下肢运动功能变化。4评估患者心肌受累程度及智能水平。结果：1临床资料：初诊DMD患者，就诊年龄10个月-16岁，平均6.61士2.68岁。≤3岁组就诊原因主要为高肌酸激酶血症、阳性家族史；4-5岁组多以走路姿势异常就诊，6-8岁组以易摔倒、蹲起困难为主要就诊原因。发现阳性家族史10例，其余为散发病例。肌电图上、下肢均呈典型肌源性损害，近端肌为重。2血肌酸激酶：初诊患者血CK均显著升高，在≤3岁、5岁和8岁分别出现3个高峰，平均CK峰值≤3岁组>5岁组>8岁组，8岁后随年龄增长CK水平反而逐年下降。地塞米松治疗后CK均大幅度下降，继续醋酸泼尼松维持治疗1月后血CK明显复升。3下肢运动功能：50例患者醋酸泼尼松治疗后运动能力较治疗前改善（蹲起P=0.032，上下楼梯P=0.014）,包括24例接受间断静脉激素治疗患者。4心肌核素显像：37例心肌显像示心室肌放射性核素分布明显不均匀，呈“花斑样”改变，21例轻度受损，9例中度受损，7例重度受损，其中下间隔（14例）受累最多，前间隔受累最少（3例），心肌受损程度与年龄成正相关（P<0.01，rs=0.685）。5智能评估：24例DMD患者言语、操作能力及总体智商水平均较同年龄儿童低下（P<0.01），言语及操作能力发育无显著性差异（P=0.259>0.05），二者发育平衡。结论：1DMD为发病率最高的进行性肌营养不良，6-7岁为首诊高峰年龄组，多以走路姿势异常为首发症状，随病情进展出现易摔倒、跑步慢及上下楼梯、蹲起困难等临床表现。2DMD患者亚临床阶段即存在高CK血症、心肌损害，心肌损害程度与年龄成正相关；DMD患者存在智能损害，总体智商水平低下。3上述激素治疗后患者下肢运动功能较前改善，静脉激素可短时间降低血CK水平，口服小剂量激素不能降低血CK水平。4糖皮质激素治疗对维持DMD患者运动功能、延长行走时间、保护心功能有效。

    Dystrophinopathy is a group of inherited diseases caused by the defect ofdystrophin protein. All of these diseases are caused by mutations in the DMDgene at Xp21.2which encodes the protein dystrophin. Both male and femalecan have symptom, but male patients accounted for the vast majority of. Thedystrophinopathy spectrum include: Duchenne muscular dystrophy (DMD),Becker muscular dystrophy (BMD)(inclusive of asymptomatic hyperCKemia,cramps and myalgia, quadriceps myopathy), X-linked dilated cardiomyopathy(XLDCM), and manifesting/nonmanifesting DMD/BMD carriers. DMD is themost common and severe type of the dystrophinopathies. The disease onsets at5-6year old presented with walking slowly, easy to fall and abnormal walking,followed by progressive loss of lower leg muscle strength and ambulation,loss of ambulation at10-12year old, ultimately succumb to restrictive lungdisease or cardiac death at20-30year old. Patients with BMD present laterthan those with DMD and they ambulate independently until at least16yearsof age, with a mean age of30years. Progression is slower and there is alonger life expectancy. Carriers and females with contiguous gene deletionsyndromes may exhibit the entire spectrum of muscle weakness, from a fullDuchenne phenotype to mild proximal muscle weakness, calf hypertrophywith mild pathological findings in muscle, to essentially normal musclefunction.The DMD gene remains the largest gene yet discovered, contains79exons, and2.2million base pairs of genomic DNA encodes dystrophin protein.The dystrophin protein has four distinct domains. The amino-terminusassociates with actin or an actinlike protein. The rod domain has long flexiblerows of24spectrin-like a-helical repeats. There is a cysteinerich region, andfinally a unique carboxy-terminus. The dystrophin protein links thecytoskeleton to the basal lamina. It links with dystroglycan and sarcoglycans to form dystrophin glycoprotein complex (DGC) and to maintain themembrane constancy. Mutations lead to breakdown of the entire criticalcomplex, resulting in fragility of the sarcolemma. Intense muscle contractionintensifies this damage and leads to calcium influx and accelerated damage tomuscle fibers.In this research, we selected patients as followed from skeletalmuscle genetic resources specimens library:1) molecular pathological andgenetic analysis of some patients with dystrophinopathy, then choose twofemale MCs families to research their clinical and genetic features;2) lowerlimb skeletal muscle MRI study of42patients with confirmeddystrophinopathy diagnosis;3) glucocorticoid treatment analysis of DMDpatients. We did clinical, pathological and molecular analysis among thesepatients in order to obtain the clinical, pathological, molecular character andobservation of glucocorticoid treatment in dystrophinopathy.Part1Molecular biological study of DystrophinopathyObjective: Dystrophinopathy is a group of inherited muscular disorderscaused by disfunction of dystrophin. The dystrophinopathy spectrum include:Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD),X-linked dilated cardiomyopathy (XLDCM), and manifesting/nonmanifestingDMD/BMD carriers. Most heterozygous female carriers of DMD mutationsare asymptomatic; however, between8and22%of these carriers aremanifesting carriers (MCs). The pathogenesy of female MCs is unclear bynow, skewed X chromosome inactivation(XCI) pattern is the hot spot ofpresent studies. Beause the number of female carrier is little, the relationshipof gene mutation, XCI and clinical manifestation is indistinct. In this research,we studied the the clinical, muscle pathological and genetic data ofdystrophinopathy and two female carrier families, to investigate the charactersand possible pathogenesy of female carrier.Methods:1Inclusion criteria of selected patients:1) Disease onset at juvenile; weaknessand atrophy at proximal muscles, calf pseudohypertrophy, Gower’s sign(+);2) serum creatine kinase increased; electrophysiology study showed myogenicchanges;3) Histochemical stains of biopsied muscle specimen showedmuscular atrophic change; Anti--dystrophin-N，-C，-R monoclon antibodyimmunohistochemical stains showed dystrophin protein absolutely absent ordecreased severely.2Histochemical and immunohistochemical stains of muscle biopsy: All theselected patients underwent open biopsy of biceps brachii, histochemical andanti-dystrophin-N、-C、 R immunohistochemical stains of sample andpathologic analysis.3DMD gene sequencing: Multiplex PCR and denaturing high performanceliquid chromatography (DHPLC) were used to detect exon deletion andduplication mutation. Direct sequencing was used to detect point mutation.4Clinical and genetic analysis of two female MCs and families4.1Family data: selected two families including female MCs with mucleweakness and wasting of proximal limb, and male patients of DMD/BMD.4.2The proband and relatives of family underwent ECG, myocardial perfusionimaging and muscle MRI of lower limb to assess cardial conduction,myocardial perfusion, left ventricular function and muscle involvement；4.3Histochemical and immunohistochemical stains of muscle biopsy: asabove.4.4Karyotype analysis: Female MCs and female asymptomatic carriersunderwent conventional GRQ karyotype analysis.4.5DMD gene sequencing: as above.4.6XCI analysis: Methylation of the highly polymorphic HpaII restrictionendonuclease site in the androgenreceptor(AR) locus correlates with XCI. Weused HpaII digestion followed by PCR to determine the methylation status ofboth the maternal and paternal X chromosomes. Alleles that are active will bedigested while the inactive alleles are not. The ratio of undigested parentalalleles gives the pattern of inactivation.Results:1Among80dystrophinopathy patients, there were67DMDs,10BMDs,2 female MCs,1asymptomatic hyperCKemia.2Biopsied muscle pathology: Histochemical stains of biopsied musclespecimen showed muscular atrophic change; Anti--dystrophin-N，-C，-Rmonoclon antibody immunohistochemical stains showed dystrophin proteinabsolutely absent(67cases) or decreased(13cases).3DMD gene sequencing: We did gene sequence in14patients,1patient withBMD found no pathogenic variations,1DMD found point mutationc.4178T>A,12patents found exon deletion.4Female MCs study4.1Clinical manifestation: Both two families were X-linked recessiveinherited, two female MCs had different degree of muscle weakness andwasting of proximal limb, without calf pseudohypertrophy. Proband of family1had lightly weakness and late onset age, while proband of family2hadmarkedly weakness and early onset age.4.2Cardiac involvement of patients: The ECG and myocardial perfusionimaging of proband of family1were normal. Myocardial perfusion imagingof proband of family2showed decreased perfusion in cardiac apex, ejectionfraction was70%, left ventricular structure and function were normal.4.3Muscle MRI: Proband of family1: asymmetric muscle involvement,selected involvement was silmilar as DMD; proband of family2: almost allmuscles were severely damaged and replaced by connective or fat tissue, onlygracilis, extensor hallucis longus and flexor digitorum longuswere relativelypreserved; Ⅳ-4of two family2: selected involvement was silmilar as BMD.4.4Histochemical and immunohistochemical analysis of muscle biopsy: twoproband and Ⅳ-4of two family2had muscular dystrophic changed inhistochemical stains, immunohistochemical stains showed dystrophinabsent/decreased in some sarcolemma, a mosaic pattern of dystrophinexpression was seen in proband of family1, while dystrophin-N,-R wasabsent and-C was decreased in two patients of family2.4.5Karyotype analysis: Proband of two families and Ⅱ-5、Ⅲ-7of family2were normal female karyotype. 4.6DMD gene sequencing: DMD gene exon61heterozygous deletion wasfound in proband and Ⅱ-7of family1. DMD gene exon12-43homozygousdeletion was found in proband, Ⅱ-5and Ⅲ-7of family2, heterozygousdeletion was found in Ⅳ-4of family2. Point mutation was not found.4.7XCI analysis: Proband of two families and Ⅲ-7of family2wereuninformative.Conclusions:1Histochemical and immunohistochemical stain with anti-dystrophinmonoclonal antibody of skeletal muscle is an important method to clinicalscreens for dystrophinopathy, guide gene sequencing and determine clinicalphenotype and prognosis.2Most of dystrophinopathy have DMD gene deletion/duplication,deletion/duplication muation should be conducted first, then screen pointmutation.3The clinical manifestation degree of female MCs is related with age ofonset, the clinical symptom is corresponding with histochemical expression ofskeletal muscle biopsy, but is independent with dystrophin protein expressionin immunohistochemical stain.4Both two families have exon deletion of DMD gene, XCI analysissometimes is necessary. The relationship between clinical phenotype and genemutation in male patients consists with the reading frame theory, but femaledon’t.Part2The lower limber muscle MRI study of dystrophinopathyObjective: The dystrophinopathy spectrum include: Duchenne musculardystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilatedcardiomyopathy (XLDCM), and manifesting/nonmanifesting DMD/BMDcarriers. The pathogenesy is the absence of dystrophin, as a cytoskeletalprotein, results in structural fragility, membrane permeability, metabolic crisis,and progressive myocyte degeneration, necrosis and regeneration, connectivetissue markedly increased. Over time, the supply of regeneration cells isexhausted and damage to muscle fibers progresses, the number of muscle cells per unit volum reduced, replacement by connective tissue and fat contributesin part. Research has indicated that increased signal intensity on T1imagesmay reflect increased fibrous tissue or adipose tissue, and fat suppressionimaging showed decreased signal. Few pelvis and thigh MR imaging studiesin DMD patients have shown a characteristic pattern of fatty infiltration thatspares the gracilis, sartorius, and semimembranosus muscles, the quadricepsfemoris, gastrocnemius and soleus muscles are predominantly affected,peroneal muscles is a characteristic feature of boys with DMD.We analysed the clinical, laboratory, and MRI data of42patients withdystrophinopathy who visited our department in2004-2012, to summarymuscle injury, the relationship between muscle choice and the muscle strength,and to investigate diagnosis and different diagnosis point at molecular imaginglevel.Methods:1Inclusion criteria of selected patients:1) onset at teenage，with a historyof progressive muscle weakness, calf pseudohypertrophy, Gower sign(+);2)significantly elevated serum creatine kinase levels, myogenic change inelectromyogram;3) Muscle biopsy showed muscular dystrophy pathologicchanges, In anti-dystrophin-N,-C, and-R monoclonal antibodyimmunohistochemical staining, dystrophin complete absence in DMD andpartial absence in BMD.2Summarized analysis of42cases of patients enrolled in clinicaldata, including: sex, onset age, blood CK, semi-quantitative assess andstatistical analysis proximal and distal muscle strength and muscle injury inthigh and lower leg MRI.3Analysis and discuss the relationship of the clinical data and lowerextremity muscle injury in MRI of dystrohpinopathy.Results:1Clinical findings: There were33cases of DMD,7case of BMD,1caseof asymptomatic hyperCKemia and one female carrier. Thirty-three boys withDMD between2and12years of age participated in the study. Negative correlations existed between proximal and distal muscle strength and patientage (proximal: r=-0.550, P=0.00<0.05; distal r=-0.400, P=0.021<0.05). Thatmeans muscle strength is getting worse with age. There was a significantpositive correlation between proximal muscle and distal muscle strength(r=0.642, P <0.01).2Muscle MRI findings: The MRI changes in the thigh were more severethan the lower leg (P=0.000<0.001). In addition, the anterior group was moresevere than posterior group in thigh, whereas in the lower leg, the posteriorwas more severe than the anterior. Of the thigh muscles, the vastus lateraliswas the most involved, and the gracilis was the least involved. In the lower legmuscles, the long fibular muscle was the most involved, and the anterior tibialmuscle was least involved. BMD patients had similar characteristics in muscleMRI as DMD patients. The patient with asymptomatic hyperCKemia and theDMD/BMD carrier were slightly affected in the quadriceps femoris andgastrocnemius, but the rest of their muscles were well preserved.3Relationship between muscle MRI and clinical data: There was nostatistically significant correlation between serum CK levels and MRI muscleinjury scores (P=0.266). There was a significant positive correlation betweenthe MRI muscle injury scores in the thigh and lower leg and patient age (thigh:r=0.720, P=0.009; lower leg: r=0.680, P <0.01). There was a strong negativecorrelation between proximal and distal muscle strength compared with thedegree of muscle injury in the thigh and lower leg in MRI analyses (thigh:r=-0.611, P <0.01; lower leg: r=-0.462, P=0.012).Conclusions:1The lower limb MRI showed selective muscle involvement indystrophinopathy, the selective involvement character was corresponding withclinical manifest. The mucle injury even can appear in subclinical period.2Muscle involvement of dystrophinopathy in MRI is regular, whichcontribute to diagnose dystrophinopathy at molecular imaging level and isessential for the diagnosis and differential diagnosis of progress musculardystrophy, also can guide mutate gene sequencing. 3The severity of muscle involvement in MRI can reflect clinical severity,that is important for pathogenetic condition evaluation, rehabilitativetreatment instruction and curative effect evaluation.Part3The study of glucocorticoid treatment of DMD patientsObjective: DMD is the most common and severe type of thedystrophinopathies, loss of ambulation at10-12year old, ultimately succumbto restrictive lung disease or cardiac death at20-30year old, with significantlyelevated serum creatine kinase levels, myogenic change in electromyogram.Currently, there is no effective cure for DMD, mainly to savethe patient’s motor function, prevention of complications for treatmentpurposes, including glucocorticoid treatment, appropriate rehabilitationexercises and orthopedic management and so on. Number of large-scaleclinical trials had shown that glucocorticoid treatment can increase musclestrength in short term,and slow down the rate of loss of muscle strength,preservation movement function and improve the quality of life. Retrospectiveanalysis the data of96cases of DMD to investigate retrospectively the clinicaland laboratory appearance of DMD and evaluate therapeutic efficacy ofglucocorticoid.Methods:1Inclusion criteria of selected patients: Progressive muscle wasting andweakness of lower limb, calf pseudohypertrophy; significantly elevated serumcreatine kinase levels, myogenic change in electromyogram; biceps brachiibiopsy showed muscular dystrophy pathologic changes, In anti-dystrophin-N,-C, and-R monoclonal antibody immunohistochemical staining showeddystrophin complete absence in sarcolemma.2Grouping and glucocorticoid therapy: The patients was grouped base onage of first diagnosis. All the patients were given dexamethasone5-10mgintravenous infusion for10-15days base on weight, age and serum CK level,then rechecked serum CK and given oral prednisone0.5-0.75mg/kg.dmaintenance treatment continuely(prednisone was given as0.75mg/kg.d topatients≤8year old, gradually with the weight gain and up to the dosage of 30mg,0.5mg/kg.d was given to patients>8year old), rechecked serum CKone month later. All the patients accepted glucocorticoid therapy signed aninformed consent.3Statistical analysis the serum CK and lower limb motor funcion beforeand after glucocorticoid therapy.4Assess the myocardiac invlovement and intelligent level of DMDpatients.Results:1Clinical findings: The age of DMD patients visited hospital first were10month to16year old(mean6.61士2.68year). The reasons that patient visithospital were: hyperCKemia and family medical history in≤3year old group;abnormal walking posture in4-5year group; easy to fall and squat difficultiesin6-8year group. In EMG myogenic changes were found in upper and lowerlimbs, espically in proximal muscles.2Serum CK: The level of serum CK had three peaks at the≤3years,5years and8years respectively,and significantly decreased after10-15days’dexamethasone(5-10mg) intervenous drop infusion, and increased againafter1month’s prednisone acetate(0.5-0.75mg/kg.d) oral administration. Theserum CK level of24cases who accepted glucocorticoid therapy repeatedlydecreaed after dexamethasone intervenous drop infusion, increased one monthlater.3Lower limb motor ability: The motor ability improved in50cases ofDMD patients with long-term oral prednisone(P=0.032, P=0.014), including24cases receiving intravenous dexamethasone intermittently.4Radionuclide imaging of cardiac muscle: The myocardial perfusionimaging of37cases of DMD showed significantly uneven ventricularradionuclide distribution, was “spotted like” change. There were21casesslightly injuried,9cases moderately injuried and7cases markedly injuried.The infer-septa(14cases) was most involved and anterior septa(3cases) wasinvolved least. There was positive relation between cardiac injury and age（P<0.01，rs=0.685）. 5Intelligence assessment: The speech and operate ability, intelligencequotients in24DMD patients were lower than normal population（P<0.01）.There was not significant difference between speech and operate ability（P=0.259>0.05）.Conclusions:1DMD was the most common type of progressive muscular dystrophy,patients usually visit hospital at6-7year old, onset with abnormal walkingposture, followed with progressive lower limb weakness.2There are high CK hyperlipidemia and myocardial damage in thesubclinical stage of DMD, myocardium impairments are positively correlatedwith age. The patient have intelligence involvement, intelligence quotientswere lower than normal population.3Glucocorticoid treatment can increase muscle strength in short term andglucocorticoid intervenous drop infusion reduced the serum CK level, but oralglucocorticoid can not reduced the serum CK level.4Glucocorticosteroid therapy has an important effect on the protection ofmotor and cardiac funcions.

Dystrophinopathy分子生物学及临床研究

中文摘要5-12ABSTRACT12-21英文缩写22-23引言23-25第一部分 Dystrophinopathy 分子生物学研究25-50    前言25-26    材料与方法26-29    结果29-32    附图32-42    附表42-43    讨论43-46    小结46    参考文献46-50第二部分 Dystrophinopathy 下肢骨骼肌 MRI 研究50-63    前言50    材料与方法50-52    结果52-54    附图54-57    讨论57-59    小结59-60    参考文献60-63第三部分 杜兴型肌营养不良糖皮质激素治疗分析63-76    前言63    材料与方法63-65    结果65-67    附图67-69    附表69-70    讨论70-73    小结73    参考文献73-76结论76-77综述一 Dystrophinopathy 研究进展77-98    参考文献91-98综述二 遗传性骨骼肌疾病下肢骨骼肌MRI研究进展98-111    参考文献107-111致谢111-113个人简历113-114

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