本文關(guān)鍵詞：Dystrophinopathy分子生物學(xué)及臨床研究，由筆耕文化傳播整理發(fā)布。

        DMD基因變異導(dǎo)致的dystrophin蛋白缺陷引起的一組遺傳性疾病稱為dystrophinopathy。DMD基因定位在Xp21.2，編碼蛋白dystrophin。男女均可發(fā)病，但男性患者占絕大多數(shù)，臨床表型包括：杜興型肌營養(yǎng)不良（Duchenne muscular dystrophy, DMD）、貝克型肌營養(yǎng)不良（Beckermuscular dystrophy, BMD）（包括無癥狀高肌酸激酶血癥、肌痙攣/肌痛、股四頭肌肌�。�、X連鎖擴張型心肌�。╔-linked dilated cardiomyopathy,XLDCM）、有/無癥狀DMD基因變異攜帶者。其中DMD是發(fā)病率最高、臨床表現(xiàn)最重類型，多于5-6歲起病，表現(xiàn)走路慢，易摔倒，走路姿勢異常，進(jìn)展加重出現(xiàn)明顯雙下肢、腰帶肌無力，蹲下站起、上下樓梯費力，約10-12歲左右不能行走，20-30歲死于心肺功能衰竭。BMD臨床癥狀出現(xiàn)較DMD晚，可保持行走能力到16歲，病情進(jìn)展相對緩慢，壽命較長。女性DMD基因缺陷攜帶者和鄰近基因缺失女性患者臨床表現(xiàn)差異巨大，重者可為典型DMD表現(xiàn)，輕者表現(xiàn)為輕度近端肌無力、腓腸肌假性肥大，甚至肌肉功能基本正常。Dystrophinopathy致病基因DMD至今仍是已發(fā)現(xiàn)的最大的基因，包含79個外顯子，編碼蛋白dystrophin，dystrophin蛋白包括4個不同的區(qū)域，氨基端與肌動蛋白（actin）或其相關(guān)蛋白相連，棒狀端擁有長的24個膜收縮蛋白樣（spectrin-like）重復(fù)區(qū)和位于兩端的4個絞鏈區(qū)，胱氨酸富集區(qū)和羧基段選擇性剪接區(qū)。Dystrophin蛋白連接細(xì)胞骨架與基底膜，并與其他蛋白（dystroglycan、sarcoglycan）形成肌營養(yǎng)不良糖蛋白復(fù)合體（dystrophinglycoprotein complex, DGC）維持細(xì)胞膜穩(wěn)定性�；蜃儺悓�(dǎo)致整個蛋白復(fù)合體破壞使肌細(xì)胞膜脆性增加、穩(wěn)定性破壞，強烈的肌肉收縮加劇肌細(xì)胞膜破壞使鈣離子內(nèi)流加速肌纖維的破壞。本研究從骨骼肌遺傳資源標(biāo)本庫中篩選：1）部分dystrophinopathy患者進(jìn)行分子病理和基因測序，重點選擇2例有癥狀女性DMD基因變異攜帶者家系進(jìn)行臨床和基因變異分析；2）確診的42例dystrophinopathy患者進(jìn)行下肢骨骼肌MRI研究分析；3）DMD患者激素治療分析，旨在對dystrophinopathy進(jìn)行臨床、影像、基因和激素治療系列研究，發(fā)現(xiàn)臨床、病理及分子生物學(xué)特點及治療觀察經(jīng)驗。第一部分Dystrophinopathy分子生物學(xué)研究目的：Dystrophinopathy是dystrophin蛋白結(jié)構(gòu)、功能異常相關(guān)的一組遺傳性骨骼肌疾病，致病基因DMD定位在Xp21.2，包含79個外顯子，編碼蛋白dystrophin。臨床表型包括：Duchenne muscular dystroph（yDMD）、Becker muscular dystrophy（BMD）、X連鎖擴張型心肌病（X-linked dilatedcardiomyopathy，XLDCM）、有/無癥狀DMD基因變異攜帶者。約8-22%的女性DMD基因變異攜帶者出現(xiàn)程度不同的肌無力表現(xiàn)，稱為有癥狀攜帶者（manifesting carriers, MCs）。目前女性MCs發(fā)病機制尚未完全明確，傾斜的X染色體失活模式是目前研究熱點。因病例較少尚未發(fā)現(xiàn)女性MCs基因熱點突變，基因變異-X染色體失活-臨床表現(xiàn)關(guān)系不明。本文對dystrophinopathy患者和2例女性MCs家系進(jìn)行臨床、骨骼肌病理和基因變異分析，旨在發(fā)現(xiàn)dystrophinopathy基因變異特點，重點探討女性MCs的臨床、基因變異特點及其可能的發(fā)病機制。方法：1臨床病例入選標(biāo)準(zhǔn)：1）臨床表現(xiàn)：緩慢進(jìn)行性加重四肢近端肌、腰帶肌無力、萎縮，腓腸肌假性肥大，Gower征（+）；2）高CK血癥，肌電圖呈肌源性損害；3）活檢骨骼肌組織化學(xué)染色呈肌營養(yǎng)不良改變，抗-dystrophin-N，-C，-R單克隆抗體免疫組織化學(xué)染色dystrophin蛋白完全/部分缺失。2活檢骨骼肌病理分析：入選病例行骨骼肌活檢組織化學(xué)染色和抗dystrophin-N、-C、R單克隆抗體免疫組織化學(xué)染色病理分析。3DMD基因測序：多重PCR-DHPLC檢測外顯子缺失和重復(fù)突變，直接測序檢測點突變。4女性MCs2例及家系臨床和基因分析：4.1家系資料：選取有肢體近端肌無力、萎縮的2例女性患者，并且家族中有男性DMD/BMD患者的家系。4.2對家系患者及部分親屬行心電圖、心肌核素顯像及下肢肌肉MRI檢查，評價心肌、骨骼肌受累情況。4.3骨骼肌活檢組化和免疫組化染色：家系1先證者及家系2先證者及Ⅳ-4行開放式左肱二頭肌活檢病理分析（方法同上）。4.4核型分析：女性MCs和女性無癥狀攜帶者做常規(guī)GRQ帶顯色核型分析。4.5DMD基因測序：方法同上。4.6X染色體失活（X chromosome inactivation，XCI）分析：利用患者外周血DNA的擴增雄激素受體（androgen receptor，AR）區(qū)域，根據(jù)等位基因AR區(qū)域CpG數(shù)量不同，可區(qū)分父母X染色體。HpaII限制性內(nèi)切酶消化作用確定其父母雙方的X染色體的活性。結(jié)果：1臨床資料：80例dystrophinopathy患者中，DMD患者67例，BMD患者10例，女性MCs2例，無癥狀高CK血癥1例。2骨骼肌活檢組化和免疫組化病理：全部病例活檢骨骼肌組織化學(xué)染色呈肌營養(yǎng)不良改變，輕重程度與病程相關(guān)；抗dystrophin-N、-C、-R單克隆抗體免疫組織化學(xué)染色肌細(xì)胞膜dystrophin蛋白完全（67例）/部分缺失（13例）。3基因測序：14例dystrophinopathy患者行DMD基因測序分析：1例BMD未發(fā)現(xiàn)致病基因突變，1例DMD患者發(fā)現(xiàn)點突變c.4178T>A，余12例患者發(fā)現(xiàn)DMD外顯子缺失突變。4女性MCs2例及家系臨床和基因研究4.1臨床表現(xiàn)：2個家系均呈X-連鎖隱性遺傳，先證者均表現(xiàn)不同程度肢體近端肌無力，無明顯腓腸肌肥大。家系1先證者發(fā)病晚，臨床癥狀較輕，家系2先證者發(fā)病早癥狀較重。4.2家系患者心肌受累情況：家系1先證者心電圖和心肌核素顯像未見異常。家系2先證者心肌核素顯像：心尖處血流灌注輕度降低，左室射血分?jǐn)?shù)：70%，左室結(jié)構(gòu)和功能大致正常。4.3下肢肌肉MRI：家系1先證者：下肢肌肉受損不對稱，肌群受損與DMD相似。家系2先證者：僅股薄肌、伸踇長肌、屈趾長肌相對保留，剩余肌肉均被脂肪和結(jié)締組織替代。家系2Ⅳ-4肌群受損與BMD相似。4.4骨骼肌活檢組化和免疫組化病理：2個家系先證者和家系2Ⅳ-4活檢骨骼肌病理均為肌營養(yǎng)不良改變，抗dystrophin-N、-C、-R單克隆抗體免疫組織化學(xué)染色，肌纖維膜dystrophin蛋白在部分肌細(xì)胞膜上缺失/減弱，，家系1先證者肌纖維膜dystrophin蛋白部分缺失呈馬賽克樣分布；家系2兩名患者dystrophin-N、-R端完全缺失，-C表達(dá)減弱。4.5核型分析：家系1先證者和家系2先證者和Ⅱ-5、Ⅲ-7常規(guī)GRQ帶染色體核型分析為正常女性核型。4.6DMD基因測序：家系1先證者和Ⅱ-7發(fā)現(xiàn)外顯子61雜合性缺失。家系2先證者、Ⅱ-5和Ⅲ-7發(fā)現(xiàn)外顯子12-43雜合性缺失，家系2Ⅳ-4發(fā)現(xiàn)外顯子12-43純合性缺失。2例先證者未發(fā)現(xiàn)有臨床意義點突變。4.7XCI分析：家系1先證者、家系2先證者和Ⅲ-7X染色體失活檢測均不能區(qū)分父母的等位基因(uninformative)。結(jié)論：1活檢骨骼肌組織化學(xué)、抗-dystrophin單克隆抗體免疫組織化學(xué)染色病理分析有助于臨床診斷dystrophinopathy，指導(dǎo)目的基因分析，判斷臨床表型和疾病預(yù)后。2對于臨床診斷dystrophinopathy，基因分析首選片段缺失/重復(fù)篩查，再行點突變檢測。3女性MCs臨床癥狀嚴(yán)重程度與發(fā)病年齡有關(guān)，與骨骼肌病理像輕重程度相對應(yīng)，與dystrophin蛋白表達(dá)無關(guān)。4女性MCs2例及家系均DMD基因外顯子片段缺失，均未檢測到具有臨床意義點突變，女性MCs應(yīng)常規(guī)進(jìn)行核型和XCI分析；女性MCs臨床表型-基因變異不符合閱讀框?qū)W說，男性患者基因變異-臨床表型多數(shù)符合閱讀框?qū)W說。第二部分Dystrophinopathy下肢骨骼肌MRI研究目的：Dystrophinopathy主要發(fā)病機制為肌細(xì)胞骨架蛋白dystrophin缺失、肌細(xì)胞穩(wěn)定性破壞，持續(xù)肌細(xì)胞變性、壞死和再生，結(jié)締組織增生，隨病程進(jìn)展，單位容積肌細(xì)胞數(shù)目減少，脂肪組織替代。研究發(fā)現(xiàn)DMD患者肌肉損害T1加權(quán)成像表現(xiàn)為肌肉組織中出現(xiàn)短T1信號，壓脂呈低信號，說明肌肉組織間脂肪組織浸潤，隨病情進(jìn)展脂肪浸潤程度增加，并表現(xiàn)出特異性選擇性肌肉損害：大腿股薄肌、縫匠肌及半膜肌相對保存，股四頭肌、比目魚肌、腓腸肌損害明顯，其中腓骨肌受損是DMD肌肉損害的特征之一。分析2004-2012年我科確診的42例dystrophinopathy患者臨床、下肢骨骼肌MRI特點，旨在總結(jié)、分析選擇性肌群受累特點，探索分子影像學(xué)水平dystrophinopathy的診斷要點，及其與其它類型PMD的鑒別診斷要點。方法：1研究對象入組標(biāo)準(zhǔn)：1）兒童、青少年起病，下肢近端肌無力、肌萎縮，腓腸肌假性肥大，Gower征（+）；2）血CK顯著升高，肌電圖呈肌源性損害；3）肱二頭肌活檢組織化學(xué)染色病理分析大量的肌纖維變性、壞死和再生，結(jié)締組織增生；抗dystrophin-N，-C，-R單克隆抗體免疫組化染色，肌細(xì)胞膜dystrophin蛋白完全缺失診斷DMD，部分缺失診斷BMD。2歸納分析42例入組患者臨床資料，包括：性別、發(fā)病年齡、血肌酸激酶值，半定量評估、統(tǒng)計學(xué)分析下肢近/遠(yuǎn)端肌力，大小腿骨骼肌MRI受累肌群。3總結(jié)分析dystrophinopathy下肢骨骼肌MRI受累肌群特點及與臨床表現(xiàn)相互關(guān)系。結(jié)果：1臨床資料：下肢肌力與年齡呈負(fù)相關(guān)(大腿：r=-0.550, P=0.001<0.05,小腿：r=-0.400, P=0.021<0.05），近端肌力與遠(yuǎn)端肌力呈正相關(guān)（r=0.642,P<0.01）。2選擇性肌群受累程度分析：DMD組下肢骨骼肌MRI，大腿肌肉受累程度重于小腿（P<0.001）；大腿肌群損害出現(xiàn)早且重于小腿。大腿肌群中股薄肌受累程度最輕，半腱肌次之，前群肌中股外側(cè)肌最重；小腿肌群中脛骨前肌受累程度最小，后群肌中腓腸肌外側(cè)頭受累重于比目魚肌和腓腸肌內(nèi)側(cè)頭，前群肌中腓骨長肌受累最重。6例BMD肌群受累特點與DMD相似，1例無癥狀高CK血癥和1例DMD基因缺陷攜帶者僅在股四頭肌和腓腸肌呈輕度受累。3肌肉受累程度與臨床資料對比分析：血CK與下肢肌肉受損程度無相關(guān)性（P=0.266>0.05）。大腿肌群與小腿肌群損傷程度均與年齡存在正相關(guān)性（大腿： r=0.720，P=0.009<0.05；小腿： r=0.680，P <0.01）；下肢近端肌力與大腿肌肉損傷程度存在負(fù)相關(guān)性（r=-0.611, P <0.01），遠(yuǎn)端肌力與小腿肌肉損傷程度存在負(fù)相關(guān)性（r=-0.462，P=0.012<0.05）。結(jié)論：1Dystrophinopathy患者下肢骨骼肌MRI存在特征性選擇性肌群受累，受累特點與臨床表現(xiàn)相對應(yīng)，甚至在亞臨床期MRI即可呈現(xiàn)肌肉損害特點。2Dystrophinopathy下肢骨骼肌MRI選擇性肌群受累具有一定的規(guī)律性，此特點有助于分子影像水平診斷dystrophinopathy，鑒別診斷其他類型進(jìn)行性肌營養(yǎng)不良，提示臨床分型、指導(dǎo)致病基因測序。3下肢骨骼肌MRI可以客觀評價dystrophinopathy患者病情，根據(jù)受累肌群特點指導(dǎo)康復(fù)治療，評估藥物治療療效。第三部分杜興型肌營養(yǎng)不良糖皮質(zhì)激素治療分析目的：DMD是進(jìn)行性肌營養(yǎng)不良中發(fā)病率最高的類型。約10-12歲左右不能行走，20-30歲死于心肺功能衰竭。血肌酸激酶（creatine kinase，CK）顯著增高數(shù)十至數(shù)百倍，肌電圖呈肌源性損害。目前DMD無有效的根治方法，主要以保存患者運動功能、防治并發(fā)癥為治療目的，包括：糖皮質(zhì)激素治療、適當(dāng)康復(fù)鍛煉及外科矯形等。其中糖皮質(zhì)激素治療是國內(nèi)外公認(rèn)的可延緩患者肌力喪失速度，保存運動功能，提高生活質(zhì)量的藥物。回顧性分析96例DMD患者臨床、實驗室表現(xiàn)，評判糖皮質(zhì)激素治療效果。方法：1研究對象入組標(biāo)準(zhǔn)：診斷基于：明顯的下肢為主的肌無力、肌萎縮，血CK顯著升高，肌電圖呈肌源性損害，肱二頭肌活檢組織化學(xué)染色大量的肌纖維變性、壞死和再生，結(jié)締組織增生，抗dystrophin-N，-C，-R單克隆抗體免疫組化染色，肌細(xì)胞膜dystrophin蛋白完全缺失。2分組及激素治療方法：根據(jù)首診年齡進(jìn)行分組，再根據(jù)體重、年齡及血CK水平給予地塞米松5-10mg靜脈輸液治療10-15天復(fù)查血CK，繼續(xù)給予口服醋酸潑尼松0.5-0.75mg/kg.d維持治療，1月后復(fù)查血CK，繼續(xù)醋酸潑尼松維持治療。3統(tǒng)計學(xué)分析激素治療前后患者血CK、下肢運動功能變化。4評估患者心肌受累程度及智能水平。結(jié)果：1臨床資料：初診DMD患者，就診年齡10個月-16歲，平均6.61士2.68歲�！�3歲組就診原因主要為高肌酸激酶血癥、陽性家族史；4-5歲組多以走路姿勢異常就診，6-8歲組以易摔倒、蹲起困難為主要就診原因。發(fā)現(xiàn)陽性家族史10例，其余為散發(fā)病例。肌電圖上、下肢均呈典型肌源性損害，近端肌為重。2血肌酸激酶：初診患者血CK均顯著升高，在≤3歲、5歲和8歲分別出現(xiàn)3個高峰，平均CK峰值≤3歲組>5歲組>8歲組，8歲后隨年齡增長CK水平反而逐年下降。地塞米松治療后CK均大幅度下降，繼續(xù)醋酸潑尼松維持治療1月后血CK明顯復(fù)升。3下肢運動功能：50例患者醋酸潑尼松治療后運動能力較治療前改善（蹲起P=0.032，上下樓梯P=0.014）,包括24例接受間斷靜脈激素治療患者。4心肌核素顯像：37例心肌顯像示心室肌放射性核素分布明顯不均勻，呈“花斑樣”改變，21例輕度受損，9例中度受損，7例重度受損，其中下間隔（14例）受累最多，前間隔受累最少（3例），心肌受損程度與年齡成正相關(guān)（P<0.01，rs=0.685）。5智能評估：24例DMD患者言語、操作能力及總體智商水平均較同年齡兒童低下（P<0.01），言語及操作能力發(fā)育無顯著性差異（P=0.259>0.05），二者發(fā)育平衡。結(jié)論：1DMD為發(fā)病率最高的進(jìn)行性肌營養(yǎng)不良，6-7歲為首診高峰年齡組，多以走路姿勢異常為首發(fā)癥狀，隨病情進(jìn)展出現(xiàn)易摔倒、跑步慢及上下樓梯、蹲起困難等臨床表現(xiàn)。2DMD患者亞臨床階段即存在高CK血癥、心肌損害，心肌損害程度與年齡成正相關(guān)；DMD患者存在智能損害，總體智商水平低下。3上述激素治療后患者下肢運動功能較前改善，靜脈激素可短時間降低血CK水平，口服小劑量激素不能降低血CK水平。4糖皮質(zhì)激素治療對維持DMD患者運動功能、延長行走時間、保護心功能有效。

    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分子生物學(xué)及臨床研究

中文摘要5-12ABSTRACT12-21英文縮寫22-23引言23-25第一部分 Dystrophinopathy 分子生物學(xué)研究25-50    前言25-26    材料與方法26-29    結(jié)果29-32    附圖32-42    附表42-43    討論43-46    小結(jié)46    參考文獻(xiàn)46-50第二部分 Dystrophinopathy 下肢骨骼肌 MRI 研究50-63    前言50    材料與方法50-52    結(jié)果52-54    附圖54-57    討論57-59    小結(jié)59-60    參考文獻(xiàn)60-63第三部分 杜興型肌營養(yǎng)不良糖皮質(zhì)激素治療分析63-76    前言63    材料與方法63-65    結(jié)果65-67    附圖67-69    附表69-70    討論70-73    小結(jié)73    參考文獻(xiàn)73-76結(jié)論76-77綜述一 Dystrophinopathy 研究進(jìn)展77-98    參考文獻(xiàn)91-98綜述二 遺傳性骨骼肌疾病下肢骨骼肌MRI研究進(jìn)展98-111    參考文獻(xiàn)107-111致謝111-113個人簡歷113-114

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