林曉丹 何君潔 陳萬(wàn)全 王檸 王志強(qiáng)

·專題綜述·

面—肩—肱型肌營(yíng)養(yǎng)不良癥分子學(xué)機(jī)制研究進(jìn)展

林曉丹 何君潔 陳萬(wàn)全 王檸 王志強(qiáng)

面?肩?肱型肌營(yíng)養(yǎng)不良癥（FSHD）呈常染色體顯性遺傳，以面肌、肩胛帶肌和上臂肌群肌無(wú)力和肌萎縮發(fā)病，逐漸累及軀干肌群和下肢肌群，臨床異質(zhì)性較高，預(yù)后相對(duì)較好。臨床分型包括FSHD1型和FSHD2型，前者與4q35區(qū)域D4Z4串聯(lián)重復(fù)序列缺失有關(guān)，其上游簡(jiǎn)單序列長(zhǎng)度多態(tài)性和下游特殊等位序列4qA/4qB具有選擇致病性。4q35區(qū)域DNA低甲基化啟動(dòng)表觀遺傳效應(yīng)，使D4Z4串聯(lián)重復(fù)序列內(nèi)DUX4基因去抑制致異常表達(dá)，導(dǎo)致多種肌細(xì)胞損害效應(yīng)。后者由DNA甲基化調(diào)控基因——SMCHD1基因突變所致。支持面?肩?肱型肌營(yíng)養(yǎng)不良癥是毒性功能獲得性疾病學(xué)說(shuō)，為其治療研究提供重要靶點(diǎn)。

營(yíng)養(yǎng)不良，面肩肱型； 基因； 突變； 綜述

面?肩?肱型肌營(yíng)養(yǎng)不良癥［FSHD，在線人類孟德爾遺傳數(shù)據(jù)庫(kù)（OMIM）編號(hào)：158900］于1882年由法國(guó)神經(jīng)病學(xué)家Louis Landouzy和Joseph Dejerine首次報(bào)告，亦稱為L(zhǎng)andouzy?Dejerine型肌營(yíng)養(yǎng)不良癥［1］，是繼Duchenne型肌營(yíng)養(yǎng)不良癥（DMD）和強(qiáng)直性肌營(yíng)養(yǎng)不良癥（DM）后臨床最常見(jiàn)類型，呈常染色體顯性遺傳，發(fā)病率約為1/2萬(wàn)，20歲時(shí)外顯率達(dá)95%，30%患者為新發(fā)突變［2］。通常于青少年期發(fā)病，主要表現(xiàn)為對(duì)稱性或不對(duì)稱性肌無(wú)力和肌萎縮，累及面肌、肩胛帶肌和上臂肌群，呈現(xiàn)貓臉、魚嘴、翼狀肩胛、游離肩、衣架肩等典型外觀，逐漸向下進(jìn)展累及軀干肌群和下肢肌群。面?肩?肱型肌營(yíng)養(yǎng)不良癥患者存在高度家系間和家系內(nèi)臨床異質(zhì)性，包括無(wú)癥狀攜帶者、僅面部輕微受累者和四肢癱瘓者，病程進(jìn)展緩慢，預(yù)后相對(duì)較好，一般不直接影響壽命，約20%患者最終依靠輪椅［3］。除骨骼肌受累外，部分患者出現(xiàn)神經(jīng)性耳聾和視網(wǎng)膜毛細(xì)血管擴(kuò)張，少數(shù)嚴(yán)重患者還出現(xiàn)癲發(fā)作、智力障礙等癥狀［4?5］。面?肩?肱型肌營(yíng)養(yǎng)不良癥發(fā)病機(jī)制有別于其他單基因遺傳病，不遵循傳統(tǒng)致病性突變導(dǎo)致編碼蛋白變異的經(jīng)典模式，成為神經(jīng)肌肉病領(lǐng)域的研究難點(diǎn)。本文擬對(duì)近年來(lái)面?肩?肱型肌營(yíng)養(yǎng)不良癥相關(guān)分子學(xué)機(jī)制研究進(jìn)展進(jìn)行概述。

一、分子病理學(xué)與分子診斷

面?肩?肱型肌營(yíng)養(yǎng)不良癥的致病基因定位于第4號(hào)染色體長(zhǎng)臂亞端粒區(qū)（4q35），是首個(gè)發(fā)現(xiàn)的由大衛(wèi)星重復(fù)序列缺失導(dǎo)致的神經(jīng)系統(tǒng)遺傳性疾病，與該區(qū)域多態(tài)性EcoRⅠ片段內(nèi)長(zhǎng)度為3.30×103bp的D4Z4串聯(lián)重復(fù)序列（DRs）缺失直接相關(guān)：正常人群D4Z4基因拷貝數(shù)為11～100個(gè)；面?肩?肱型肌營(yíng)養(yǎng)不良癥患者減少至1～10個(gè)，EcoRⅠ片段長(zhǎng)度縮短至 ＜ 38 × 103bp［6］。面?肩?肱型肌營(yíng)養(yǎng)不良癥不僅基因突變類型獨(dú)特，而且存在2個(gè)少見(jiàn)的分子遺傳學(xué)現(xiàn)象：（1）10q26區(qū)域存在與4q35區(qū)域高度同源的多態(tài)性EcoRⅠ片段，此片段長(zhǎng)度亦＜38×103bp，但無(wú)致病性，二者之間存在高頻易位現(xiàn)象，應(yīng)注意鑒別［7?8］。（2）4q35 區(qū)域 D4Z4 串聯(lián)重復(fù)序列與上游 3 ×103bp處特異性簡(jiǎn)單序列長(zhǎng)度多態(tài)性（SSLP）和下游10×103bp處特殊等位序列4qA/4qB存在密切連鎖關(guān)系，D4Z4基因缺失與特異性SSLP?4qA基因型共存而致?。??10］（圖 1）。研究顯示，我國(guó)面?肩?肱型肌營(yíng)養(yǎng)不良癥的主要基因型是4A161PAS［11］。因此，大片段致病基因的分離和鑒定是基因檢測(cè)和分子學(xué)機(jī)制研究的基礎(chǔ)。采用脈沖場(chǎng)凝膠電泳（PFGE）聯(lián)合多重Southern blotting法，是目前國(guó)際指南推薦的分子診斷技術(shù)［12］。約5%面?肩?肱型肌營(yíng)養(yǎng)不良癥患者存在相應(yīng)臨床表型，未見(jiàn)4q35區(qū)域D4Z4串聯(lián)重復(fù)序列縮短，稱為面?肩?肱型肌營(yíng)養(yǎng)不良癥2型（FSHD2 型）［13］。2012 年，Lemmers等［14］采用全外顯子測(cè)序（WES）證實(shí)FSHD2型致病基因?yàn)镾MCHD1基因，該基因突變與導(dǎo)致DUX4基因表達(dá)的4qA等位基因共同作用而致病，稱為雙遺傳模式。SMCHD1基因單倍體劑量不足機(jī)制表明，SMCHD1基因突變導(dǎo)致編碼蛋白表達(dá)下調(diào)，使D4Z4串聯(lián)重復(fù)序列甲基化降低，進(jìn)而通過(guò)與面?肩?肱型肌營(yíng)養(yǎng)不良癥1型（FSHD1型）相同的表觀遺傳學(xué)機(jī)制而發(fā)揮作用，因此，不同亞型面?肩?肱型肌營(yíng)養(yǎng)不良癥具有相似的分子通路。

二、分子遺傳學(xué)及發(fā)病機(jī)制

面?肩?肱型肌營(yíng)養(yǎng)不良癥是典型人類孟德爾遺傳性疾病，但4q35區(qū)域D4Z4串聯(lián)重復(fù)序列致病性縮短的分子學(xué)機(jī)制極為復(fù)雜，一直是疾病研究的難點(diǎn)，研究者們致力于尋找D4Z4基因上下游和內(nèi)部可能的效應(yīng)基因。Gabellini等［15?16］發(fā)現(xiàn)，面?肩?肱型肌營(yíng)養(yǎng)不良癥患者4q35區(qū)域D4Z4串聯(lián)重復(fù)序列上游120×103bp處FRG1基因呈異常高表達(dá)，遂構(gòu)建轉(zhuǎn)基因小鼠模型，出現(xiàn)類似面?肩?肱型肌營(yíng)養(yǎng)不良癥臨床表型和病理改變，故認(rèn)為FRG1基因異常表達(dá)可以干擾mRNA前體剪切修飾，與骨骼肌生長(zhǎng)發(fā)育有關(guān)。因此，F(xiàn)RG1 基因成為重要候選基因［17?18］。此后多項(xiàng)研究顯示，該轉(zhuǎn)基因小鼠模型基因結(jié)構(gòu)僅與面?肩?肱型肌營(yíng)養(yǎng)不良癥患者部分相似，未能驗(yàn)證轉(zhuǎn)基因小鼠模型存在 FRG1 基因異常表達(dá)［19?20］。2010 年，Snider等［21］發(fā)現(xiàn)，4q35 區(qū)域 D4Z4 串聯(lián)重復(fù)序列內(nèi)DUX4基因表達(dá)上調(diào)，證實(shí)該基因在面?肩?肱型肌營(yíng)養(yǎng)不良癥發(fā)病機(jī)制中發(fā)揮重要作用。DUX4基因包含2個(gè)同源序列和2個(gè)富含鳥嘌呤?胞嘧啶（GC）的重復(fù)序列，形成讀碼框（ORF），其末端連接多聚腺苷酸信號(hào)（PAS）以穩(wěn)定DUX4基因轉(zhuǎn)錄和翻譯（圖1）。研究顯示，D4Z4基因富含CpG島（CpG island），CpG島主要位于轉(zhuǎn)錄調(diào)控區(qū)附近，是一種重要表觀遺傳學(xué)修飾方式［22］。2013 年，Hartweck 等［23］證實(shí)，面?肩?肱型肌營(yíng)養(yǎng)不良癥的4q35區(qū)域D4Z4串聯(lián)重復(fù)序列內(nèi)存在3個(gè)DNA低甲基化區(qū)域，即DR1、DR2和DR3區(qū)域，尤以DR1區(qū)域（位于D4Z4基因5’端）低甲基化程度最顯著。2014年，Gaillard等［24］比較面?肩?肱型肌營(yíng)養(yǎng)不良癥患者與無(wú)癥狀攜帶者DNA甲基化水平，發(fā)現(xiàn)面?肩?肱型肌營(yíng)養(yǎng)不良癥患者DNA甲基化水平明顯降低。DNA甲基化雖未改變基因結(jié)構(gòu)，但可引起局部DNA構(gòu)象穩(wěn)定性改變，從而調(diào)控基因表達(dá)，進(jìn)一步證實(shí)面?肩?肱型肌營(yíng)養(yǎng)不良癥是一種表觀遺傳效應(yīng)的遺傳性疾病學(xué)說(shuō)，DNA甲基化檢測(cè)成為參考診斷指標(biāo)［24］。

面?肩?肱型肌營(yíng)養(yǎng)不良癥具有高度臨床異質(zhì)性，可能與以下因素有關(guān)：（1）4q35區(qū)域D4Z4串聯(lián)重復(fù)序列拷貝數(shù)與臨床表型呈負(fù)相關(guān)，EcoRⅠ片段長(zhǎng)度縮短越明顯、臨床表型越嚴(yán)重、外顯年齡越早、累及肌群越多［3］。（2）表觀遺傳效應(yīng)，DNA 甲基化水平與臨床表型呈負(fù)相關(guān)，存在相同D4Z4串聯(lián)重復(fù)序列拷貝數(shù)的患者，DNA甲基化水平越低、臨床表型越嚴(yán)重［24］。（3）臨床表型的調(diào)控基因，研究顯示，SMCHD1和DNMT3B基因是FSHD1型的調(diào)控基因，SMCHD1或DNMT3B基因突變的FSHD1型患者表現(xiàn)出更嚴(yán)重的臨床表型，即SMCHD1和DNMT3B基因可能與面?肩?肱型肌營(yíng)養(yǎng)不良癥的致病性存在協(xié)同作用［25?26］。目前公認(rèn)的面?肩?肱型肌營(yíng)養(yǎng)不良癥發(fā)病機(jī)制是，4q35區(qū)域D4Z4串聯(lián)重復(fù)序列缺失致DNA甲基化水平降低，在表觀遺傳效應(yīng)調(diào)控下染色質(zhì)構(gòu)象改變失去穩(wěn)定性，引起DUX4基因在骨骼肌細(xì)胞中表達(dá)，產(chǎn)生的DUX4蛋白對(duì)肌細(xì)胞產(chǎn)生多種毒性作用。

圖1 面?肩?肱型肌營(yíng)養(yǎng)不良癥4q35區(qū)域相關(guān)基因結(jié)構(gòu)模式圖：4q35區(qū)域EcoRⅠ片段由數(shù)目不等的長(zhǎng)度為3.30×103bp的D4Z4串聯(lián)重復(fù)序列組成，其上游簡(jiǎn)單序列長(zhǎng)度多態(tài)性和下游特殊等位序列4qA/4qB具有選擇致病性，D4Z4串聯(lián)重復(fù)序列內(nèi)存在DUX4基因，其末端連接多聚腺苷酸信號(hào)Figure 1 The structure mode of FSHD related genes in 4q35 region:the fragment of 4q35?EcoRⅠ is consisted of a series of D4Z4 repeats which is composed of numbers of 3.30×103bp,its upstream SSLP and downstream specific allele sequence 4qA/4qB have pathogenic selective effect.The DUX4 gene exists inside D4Z4 repeats and connects PAS on the terminal.

三、DUX4蛋白功能研究

DUX4基因表達(dá)失調(diào)和DUX4蛋白功能異常是面?肩?肱型肌營(yíng)養(yǎng)不良癥發(fā)病的關(guān)鍵環(huán)節(jié)。DUX4基因是反轉(zhuǎn)錄基因，編碼2條全長(zhǎng)（DUX4?fl）和截短（DUX4?s）的DUX4蛋白，在人類生殖細(xì)胞和早期胚胎干細(xì)胞（ESCs）中正常表達(dá)，晚近研究顯示，DUX4基因在胚胎早期對(duì)誘導(dǎo)合子基因組激活（ZGA）起關(guān)鍵調(diào)節(jié)作用，此后則處于沉默狀態(tài)［27］，但在面?肩?肱型肌營(yíng)養(yǎng)不良癥患者骨骼肌中呈異常表達(dá)。低水平DUX4?fl蛋白可以引起下游多種改變，激活一系列去抑制級(jí)聯(lián)反應(yīng)，導(dǎo)致肌細(xì)胞凋亡和萎縮、炎癥反應(yīng)、分化缺陷和氧化應(yīng)激，但其具體生物學(xué)功能尚未完全闡明［28］。目前較為公認(rèn)的DUX4蛋白在骨骼肌中表達(dá)的病理生理學(xué)機(jī)制包括：（1）細(xì)胞凋亡學(xué)說(shuō)，DUX4蛋白可以誘導(dǎo)抑癌基因p53表達(dá)，導(dǎo)致細(xì)胞凋亡，引起肌肉損害［29］。（2）T淋巴細(xì)胞介導(dǎo)的細(xì)胞炎癥反應(yīng)學(xué)說(shuō)，DUX4蛋白異常表達(dá)可以激活免疫反應(yīng)，類似吞噬細(xì)胞介導(dǎo)的抗腫瘤反應(yīng)。免疫反應(yīng)激活CD4+T細(xì)胞和CD8+T細(xì)胞，發(fā)生以T淋巴細(xì)胞介導(dǎo)為主的血管周圍炎性細(xì)胞浸潤(rùn)，引起肌細(xì)胞肥大和細(xì)胞核聚集，導(dǎo)致肌肉損害［30］。（3）DUX4蛋白表達(dá)與長(zhǎng)末端重復(fù)序列（LTR）的反轉(zhuǎn)錄轉(zhuǎn)座子和內(nèi)源性重復(fù)序列的轉(zhuǎn)錄激活相關(guān)，同時(shí)抑制自身免疫對(duì)逆轉(zhuǎn)錄病毒感染的應(yīng)答，通過(guò)轉(zhuǎn)錄激活防御素 DEFB103 抑制肌細(xì)胞分化、再生［31］。Jones等［32］和Mitsuhashi等［33］予肌細(xì)胞、斑馬魚和蟾蜍注射微量DUX4 mRNA以建立面?肩?肱型肌營(yíng)養(yǎng)不良癥細(xì)胞和動(dòng)物模型，觸發(fā)多種級(jí)聯(lián)反應(yīng)，導(dǎo)致肌細(xì)胞凋亡、衛(wèi)星細(xì)胞發(fā)育抑制、炎癥反應(yīng)和氧化應(yīng)激反應(yīng)障礙等一系列病理生理改變。Ansseau等［34］通過(guò)顯微注射腺相關(guān)病毒載體包裝的D4Z4基因，向野生型小鼠C57BL/6導(dǎo)入不同長(zhǎng)度的D4Z4串聯(lián)重復(fù)序列，可以觀察到與人類高度同源的DUX4基因高表達(dá)，但未出現(xiàn)相應(yīng)臨床表型。因此，目前普遍認(rèn)為DUX4基因異常表達(dá)是面?肩?肱型肌營(yíng)養(yǎng)不良癥的分子學(xué)機(jī)制，但某些關(guān)鍵途徑尚不明確，如面?肩?肱型肌營(yíng)養(yǎng)不良癥是受DUX4基因異常表達(dá)的單因素調(diào)控還是多因素聯(lián)合調(diào)控。

四、小結(jié)和展望

面?肩?肱型肌營(yíng)養(yǎng)不良癥目前尚無(wú)有效治療方法，適當(dāng)?shù)目祻?fù)訓(xùn)練可以延緩疾病進(jìn)展。發(fā)病機(jī)制已趨于明朗，是表觀遺傳效應(yīng)導(dǎo)致的毒性功能獲得性（toxic gain of function）疾病，其中DUX4基因去抑制致異常表達(dá)是關(guān)鍵致病機(jī)制，成為今后治療研究的重要靶點(diǎn)?；蛑委煼矫妫饕ㄟ^(guò)反義寡核苷酸（ASO）或干擾RNA（RNAi）技術(shù)抑制DUX4基因過(guò)表達(dá)，從而達(dá)到改善肌肉損害之目的［35?36］。近年來(lái)，采用新型基因編輯技術(shù)實(shí)現(xiàn)多靶點(diǎn)精準(zhǔn)基因調(diào)控、體外調(diào)控DUX4基因表達(dá)、尋找抑制DUX4基因的小分子化合物為面?肩?肱型肌營(yíng)養(yǎng)不良癥的治療帶來(lái)新的曙光。

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Progress in researchon molecularmechanism of facioscapulohumeralmuscular dystrophy

LIN Xiao?dan,HE Jun?jie,CHEN Wan?jin,WANG Ning,WANG Zhi?qiang
Department of Neurology,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,Fujian,China
Corresponding author:WANG Zhi?qiang(Email:fmuwzq@fjmu.edu.cn)

Facioscapulohumeralmusculardystrophy (FSHD),characterized by symmetric or asymmetric muscular weakness of the initial onset of facial,shoulder?girdle and upper arm muscles,and descending to limb muscles,is a classical autosomal dominant myopathy with high clinical diversity and relatively good prognosis.FSHD is catigorized into two types,FSHD1 and FSDH2.Previous studies have demonstrated that 95%patients with FSHD1 were associated with a contraction of D4Z4 microsatellite repeats on chromosome 4q35,which was pathogenic in the genetic backgrounds,including a special sequence of simple sequence length polymorphism(SSLP)proximal to the D4Z4 repeats and the 4qA/4qB polymorphism distal to the repeats.In recent years,several reports have confirmed that 4q35 locus leads to DNA hypomethylation and inner DUX4 gene transcription by epigenetic effect.The abnormal expression of DUX4 further activates several genes,which inhibit myogenesis,sensitize cells to oxidative stress and induce muscle atrophy.And not only that,FSHD2 is formed by another methylation regulation gene——SMCHD1 mutations.More and more evidences supported that toxic gain of function mechanism plays an important role in the occurrence of FSHD.The DUX4 gene becomes an important target for treatment study in the future.

Muscular dystrophy,facioscapulohumeral; Genes; Mutation; Review

This study was supported by Key Project of the National Natural Science Foundation of China(No.U1505222),the National Natural Science Foundation of China(No.81671237),Science and Technology Plan Project of Fujian Province,China(No.2016Y9010),and Natural Science Foundation of Fujian Province,China(No.2017J01196).

10.3969/j.issn.1672?6731.2017.08.004

國(guó)家自然科學(xué)基金重點(diǎn)資助項(xiàng)目（項(xiàng)目編號(hào)：U1505222）；國(guó)家自然科學(xué)基金資助項(xiàng)目（項(xiàng)目編號(hào)：81671237）；福建省科技計(jì)劃項(xiàng)目（項(xiàng)目編號(hào)：2016Y9010）；福建省自然科學(xué)基金資助項(xiàng)目（項(xiàng)目編號(hào)：2017J01196）

350005福州，福建醫(yī)科大學(xué)附屬第一醫(yī)院神經(jīng)內(nèi)科

王志強(qiáng)（Email：fmuwzq@fjmu.edu.cn）

2017?06?05）