劉亞蘭，張華，馮永

1.中南大學(xué)湘雅醫(yī)院耳鼻咽喉頭頸外科，長沙 410008；

2.耳鼻咽喉重大疾病研究湖南省重點(diǎn)實(shí)驗(yàn)室，長沙 410008；

3.中國醫(yī)學(xué)遺傳學(xué)國家重點(diǎn)實(shí)驗(yàn)室，長沙 410078；

4.上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院耳鼻喉科，上海 200127

耳聾是人類常見高發(fā)的致殘性疾病，其中約50%的耳聾由聽覺創(chuàng)傷、耳毒性藥物聾、病毒感染、細(xì)菌感染等環(huán)境因素引起；另外 50%的先天性耳聾及臨床上原因不明的后天性耳聾由遺傳因素引起，稱為遺傳性耳聾(Hereditary hearing loss, HHL)。依據(jù)是否伴隨其他組織器官癥狀，遺傳性耳聾分為非綜合征型耳聾(Nonsyndromic hearing loss, NSHL)和綜合征型耳聾(Syndromic hearing loss, SHL)兩類，所占比例分別為 70%和 30%[1]。幾十年來，國內(nèi)外的研究小組對(duì)非綜合征型耳聾的遺傳基礎(chǔ)、發(fā)病機(jī)制及治療干預(yù)進(jìn)行了大量研究，取得了巨大的研究進(jìn)展，大量的非綜合征性耳聾致病基因被定位和克隆(已成功定位134個(gè)NSHL基因位點(diǎn)，克隆了63個(gè)NSHL致病基因(http://hereditaryhearingloss.org/)，初步建立了相應(yīng)的基因診斷技術(shù)并推廣到臨床。而大多數(shù)綜合征型耳聾發(fā)病率相對(duì)較低，全身病變的臨床表現(xiàn)變化多樣，除了聽力障礙，同時(shí)伴有眼、骨、腎、皮膚、神經(jīng)系統(tǒng)、代謝內(nèi)分泌系統(tǒng)等其他器官或系統(tǒng)的異常，種類繁多且較復(fù)雜，對(duì)病患的生存質(zhì)量影響更大。目前已報(bào)道的SHL有400多種[2]，大多數(shù)發(fā)病率低，臨床上較常見的有 Waardenburg綜合征(Waardenburg syndrome, WS)、先天性小耳畸形綜合征、前庭導(dǎo)水管擴(kuò)大綜合征(Large vestibular aquduct syndrome, LVAS)等。由于SHL臨床和遺傳異質(zhì)性極強(qiáng)，涉及的致病基因繁多，對(duì)其遺傳基礎(chǔ)及發(fā)病機(jī)制的系統(tǒng)研究變得十分困難。本課題組在前期研究WS分子機(jī)制時(shí)發(fā)現(xiàn)其致病基因 PAX3、SOX10及MITF之間存在相互作用關(guān)系。綜合征型耳聾基因涉及多個(gè)系統(tǒng)，卻可以導(dǎo)致很多共同的臨床表型，表明它們之間可能存在著潛在的共同通路和相互作用網(wǎng)絡(luò)。我們通過查閱大量文獻(xiàn)，發(fā)現(xiàn) WS和小耳畸形綜合征的發(fā)病機(jī)制理論存在共性，都是由于神經(jīng)嵴發(fā)育不全導(dǎo)致的細(xì)胞或組織的病變?；诖?，本文著眼于國際耳科研究領(lǐng)域的前沿與難點(diǎn)，通過查閱大量文獻(xiàn)并結(jié)合本研究團(tuán)隊(duì)前期的研究成果，首次針對(duì)神經(jīng)嵴發(fā)育異常導(dǎo)致相關(guān)綜合征型耳聾的共同通路和致病機(jī)制的研究進(jìn)展進(jìn)行系統(tǒng)地闡述，分析并歸納了與綜合征型耳聾發(fā)病相關(guān)的神經(jīng)嵴發(fā)育異常相關(guān)基因互作網(wǎng)絡(luò)，為系統(tǒng)地研究常見綜合征型耳聾致病基因的定位克隆以及發(fā)病機(jī)制提供研究思路和理論基礎(chǔ)。

1 神經(jīng)嵴細(xì)胞與神經(jīng)嵴病概述

神經(jīng)嵴細(xì)胞(Neural crest cells, NCC)是脊椎動(dòng)物胚胎發(fā)育過程中出現(xiàn)的一個(gè)暫時(shí)性、多潛能細(xì)胞群，起源于背神經(jīng)管的隆起——神經(jīng)嵴(Neural crest,NC)。NCC形成后向外周遷移，分化為色素細(xì)胞、交感神經(jīng)節(jié)的神經(jīng)元細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞、骨和軟骨細(xì)胞、腎上腺髓質(zhì)細(xì)胞以及平滑肌細(xì)胞等，并逐漸分化為神經(jīng)嵴源性衍生物，包括顱面部、心臟、周圍神經(jīng)系統(tǒng)等多種重要的組織結(jié)構(gòu)[3,4]。NCC的發(fā)育不全會(huì)導(dǎo)致上述細(xì)胞和組織的相關(guān)病變，統(tǒng)稱為神經(jīng)嵴病[5]，包括 WS等色素異常表型相關(guān)疾病和先天性小耳畸形等顱面部畸形相關(guān)疾病，約有20余種(表 1)。

2 神經(jīng)嵴異常參與WS和小耳畸形的發(fā)病機(jī)制

2.1 神經(jīng)嵴發(fā)育不全理論與Waardenburg綜合征

Waardenburg綜合征(WS)又稱聽力-色素綜合征，以感音神經(jīng)性耳聾和皮膚、毛發(fā)、虹膜、內(nèi)耳色素分布異常為主要臨床表現(xiàn)[6]。WS是最常見的綜合征型耳聾之一，人群發(fā)病率為1/42000，聾啞人群中發(fā)病率為0.9%～2.8%[7,8]，占先天性耳聾的2%～5%[9,10]。WS表型的不完全外顯和致病基因的多樣性導(dǎo)致其具有高度的臨床和遺傳異質(zhì)性[11～13]。依據(jù)不同的伴隨表型，可將WS分為4種亞型(WS1-4)，最常見的為 WS1和 WS2[14]。

表1 已報(bào)道的神經(jīng)嵴病

目前關(guān)于WS所具有的臨床表型的病因有多種不同假說，其中神經(jīng)嵴發(fā)育不全理論是最被認(rèn)可的一種假說[15～18]。WS以黑素細(xì)胞分化缺陷發(fā)育異常導(dǎo)致的色素分布異常和耳聾為主要特征，黑素細(xì)胞由NC分化而來，因此WS是由于NCC的發(fā)育異常導(dǎo)致的一組臨床癥候群。NCC通過增殖、遷徙、生存、分化等過程逐漸演變?yōu)楹谒厍绑w細(xì)胞—成黑素細(xì)胞后到達(dá)真皮、表皮、內(nèi)耳血管紋和眼睛的脈絡(luò)膜，并逐漸分化為黑素細(xì)胞[19～21]。黑素細(xì)胞最主要的功能是產(chǎn)生黑色素以確保毛發(fā)和皮膚的色素沉著[22～24]。與WS相關(guān)的6個(gè)基因編碼的轉(zhuǎn)錄因子(MITF、PAX3、SOX10和SNAI2)和信號(hào)分子(ENDRB和EDN3)均參與這一過程，在NC的發(fā)育中發(fā)揮重要的調(diào)控作用。不同的致病基因與不同的WS亞型相關(guān)聯(lián)，PAX3基因突變可導(dǎo)致WS1和 WS3[25,26]，MITF基因突變導(dǎo)致WS2A[27]，SNAI2基因突變導(dǎo)致 WS2D[28]，SOX10突變導(dǎo)致 WS2E[29,30]。SOX10、EDN3和 EDNRB基因突變可造成WS4[31～33]。國外已有的WS突變數(shù)據(jù)庫報(bào)道WS有近280個(gè)基因突變位點(diǎn)(http://grenada.lumc.nl/LOVD2/WS/，截至2014年6月)；而國內(nèi)相關(guān)研究[34～41]表明中國人群 WS主要致病基因?yàn)镻AX3、MITF和SOX10，已發(fā)現(xiàn)相關(guān)突變位點(diǎn)23種，目前尚無SNAI2、EDN3和EDNRB基因突變及WS3病例報(bào)道。上述WS相關(guān)基因的突變會(huì)造成NC發(fā)育異常，導(dǎo)致黑素細(xì)胞發(fā)育不良，引起黑色素合成減少，表現(xiàn)出皮膚、毛發(fā)、虹膜低色素等臨床癥狀。血管紋是內(nèi)耳耳蝸結(jié)構(gòu)之一，在內(nèi)淋巴生成過程中起重要作用。研究顯示，黑素細(xì)胞發(fā)育不良會(huì)引起血管紋中黑素細(xì)胞源性的中間細(xì)胞缺乏進(jìn)而造成柯蒂氏器(Corti)退化變性，最終導(dǎo)致感音神經(jīng)性耳聾[42]。此外，由于顱面骨、腸壁神經(jīng)節(jié)以及四肢骨骼肌肉等均為神經(jīng)嵴源性衍生物，因此 NC的發(fā)育異常必然會(huì)導(dǎo)致這些組織和器官的發(fā)育異常，從而產(chǎn)生WS的一系列伴隨癥狀。

2.2 神經(jīng)嵴細(xì)胞遷徙理論與小耳畸形

先天性小耳畸形綜合征也是常見的一類綜合征型耳聾。小耳畸形(或稱為先天性外中耳畸形)表現(xiàn)為重度耳廓發(fā)育不全，有外耳道閉鎖或狹窄，中耳甚至內(nèi)耳畸形，通常伴有聽力損失，需要耳再造術(shù)治療[43]。小耳畸形可以作為一個(gè)獨(dú)立的出生缺陷或作為綜合征的一部分[44]。流行病學(xué)調(diào)查顯示，全球小耳無耳畸形總發(fā)病率為 2.06/10000，小耳畸形為1.55/10000，無耳畸形為0.36/10000。由于入選標(biāo)準(zhǔn)的差異，不同地區(qū)的流行病學(xué)調(diào)查結(jié)果具有一定差異。意大利、法國、瑞典、芬蘭和美國的流行病學(xué)調(diào)查結(jié)果顯示，小耳畸形的發(fā)病率范圍為 0.83～4.34/10000，而西班牙、亞洲、美洲印第安和安第斯的發(fā)病率被認(rèn)為更高[43,45～49]。在我國，研究顯示先天性小耳畸形的發(fā)病率約為 1.40/10000，其中無耳為0.55/10000，小耳為0.85/10000，城鎮(zhèn)發(fā)病率高于農(nóng)村。全國以新疆發(fā)病率最高，為 2.08/10000。男女發(fā)病比例約為 2:1，右側(cè)畸形較多，雙側(cè)者約10%[50,51]。目前，國內(nèi)外已經(jīng)報(bào)道的小耳畸形相關(guān)綜合征大約有 20余種，其中常見的有 Treacher Collins綜合征、Branchiooto-Renal綜合征、Goldenhar綜合征、Nager綜合征及Miller綜合征等。相關(guān)研究已鑒定的小耳畸形相關(guān)綜合征的致病基因大約有 20余個(gè)(表 2)。

目前對(duì)先天性小耳畸形的病因和表型變異性還知之甚少，然而，發(fā)育生物學(xué)和遺傳學(xué)研究表明，神經(jīng)嵴細(xì)胞功能缺陷與小耳畸形等多種顱面綜合征相關(guān)。Luquetti等[43]認(rèn)為神經(jīng)嵴細(xì)胞紊亂和血管破壞可能為小耳畸形的病因。NCC可分成顱神經(jīng)嵴細(xì)胞(Cranial neural crest cell, CNCC)、心神經(jīng)嵴細(xì)胞(Cardiac neural crest cells)、迷走神經(jīng)嵴細(xì)胞(Vagal neural crest cells)以及體神經(jīng)嵴細(xì)胞(Trunk neural crest cells)4個(gè)主要的軸向細(xì)胞群，每一部分都沿特定路徑遷移并分化成特定的細(xì)胞[52]。其中，顱神經(jīng)嵴細(xì)胞在耳部發(fā)育中起重要作用。顱神經(jīng)嵴細(xì)胞是起源于背側(cè)神經(jīng)管的一個(gè)暫時(shí)性、可遷移、多潛能的細(xì)胞群，可以進(jìn)一步分化為前腦(Forebrain)、中腦(Midbrain)、后腦(Hindbrain)，其中，后腦的CNCC進(jìn)一步分區(qū)形成數(shù)個(gè)菱腦原節(jié)(Rhombomere′r)，這一結(jié)構(gòu)在顱頜面部的發(fā)育中起重要作用[53]。菱腦原節(jié)不僅為 CNCC 形成數(shù)支彼此分隔的遷移流提供了解剖結(jié)構(gòu)基礎(chǔ)，而且防止了顱頜面部神經(jīng)和骨等結(jié)構(gòu)的發(fā)育發(fā)生位置錯(cuò)亂。CNCC在遷移時(shí)，不是以整體的細(xì)胞團(tuán)形式進(jìn)行遷移，而是被“NCC缺失區(qū)”分割成若干特定的細(xì)胞支流，在各種微環(huán)境的誘導(dǎo)下，沿著特定的精確路線遷移至頜面部的特定部位，進(jìn)一步分化為顱頜面部的軟硬組織結(jié)構(gòu)(圖 1)。其中，耳廓起源于胚胎第一鰓弓(下頜弓)和第二鰓弓(舌骨弓)[54]。在耳廓的發(fā)育階段，胚胎受到遺傳或外界因素影響，容易出現(xiàn)耳廓的多種發(fā)育畸形。小耳畸形就是源于胚胎期第一、二鰓弓、第一咽囊、第一鰓裂和顳骨原基的顱骨結(jié)構(gòu)發(fā)育不良的一組先天性畸形。研究發(fā)現(xiàn)，在小鼠中耳廓發(fā)育期鐙骨動(dòng)脈破裂會(huì)引起小耳畸形，使CNCC不具有遷移性[55]，而CNCC能否正確遷移到指定位置，是顱頜面結(jié)構(gòu)正常發(fā)育的前提，也是各種頜面部畸形發(fā)生的重要原因。

表2 已經(jīng)鑒定的小耳畸形相關(guān)綜合征致病基因信息

圖1 神經(jīng)嵴細(xì)胞分化發(fā)育至耳的過程示意圖

3 以PAX3和SOX10為中心的神經(jīng)嵴遷移分化相關(guān)基因互作網(wǎng)絡(luò)與SHL

神經(jīng)嵴細(xì)胞功能異常不僅能導(dǎo)致神經(jīng)嵴起源的黑素細(xì)胞缺失而造成的WS，還與包括小耳畸形在內(nèi)的多種顱面綜合征相關(guān)，表明 WS和小耳畸形綜合征的發(fā)病機(jī)制理論存在共性。本課題組前期對(duì) WS分子機(jī)制進(jìn)行研究時(shí)發(fā)現(xiàn)，其致病基因PAX3、SOX10及 MITF之間存在相互作用關(guān)系[56,57]；以此理論為依據(jù)，通過查閱大量相關(guān)文獻(xiàn)發(fā)現(xiàn)有多種基因和細(xì)胞因子共同參與神經(jīng)嵴細(xì)胞不同階段遷移的調(diào)控，與神經(jīng)嵴發(fā)展相關(guān)的基因(如PAX3、SOX10、MITF、HOX、EGR2、EYA1、SALL1和SIX1等)相互之間存在一定聯(lián)系，并參與WS等色素異常表型相關(guān)SHL和小耳畸形表型相關(guān)綜合征的致病機(jī)制。

3.1 SOX10與PAX3基因在神經(jīng)嵴細(xì)胞遷移分化中的作用

SOX10與 PAX3基因編碼的蛋白不僅是具有特殊結(jié)構(gòu)的轉(zhuǎn)錄因子，還是起正調(diào)控作用的反式作用因子，通過DNA結(jié)合域直接或間接結(jié)合靶基因的上游啟動(dòng)子順時(shí)作用元件來調(diào)控靶基因表達(dá)。缺失DNA結(jié)合域的SOX10與PAX3突變蛋白由于不能與DNA結(jié)合，導(dǎo)致靶基因轉(zhuǎn)錄水平下降而不表達(dá)。研究表明，SOX10與 PAX3基因在神經(jīng)嵴細(xì)胞的遷移和分化中起到了十分重要的作用[58]。

配對(duì)盒基因(Pair box 3，PAX3)位于染色體2q35-2q37，編碼的蛋白是高度保守的轉(zhuǎn)錄因子，由479個(gè)氨基酸組成，含有 4個(gè)高度保守的結(jié)構(gòu)域，分別為配對(duì)盒結(jié)構(gòu)域(Paired box domain, PD)、同源結(jié)構(gòu)域(Homeodomain, HD)、鋅肽序列和轉(zhuǎn)錄激活域(TA)。其中 PD在 PAX3的結(jié)構(gòu)和功能中起重要作用，PD通過與SOX10的HMG相互作用以調(diào)控PAX3活性[59]，而且PAX3必須通過PD與SOX10結(jié)合才能協(xié)同激活c-RET和MITF的轉(zhuǎn)錄[60,61]。此外，大多數(shù)PAX3突變都發(fā)生于含有部分PD結(jié)構(gòu)域的2號(hào)外顯子上，這些無義或移碼突變由于影響了PD結(jié)構(gòu)域與DNA的緊密結(jié)合而導(dǎo)致PAX3的功能異常。PAX3突變可導(dǎo)致心臟神經(jīng)嵴功能異常、神經(jīng)嵴源性細(xì)胞組織功能不良、神經(jīng)管畸形、中樞神經(jīng)系統(tǒng)異常以及四肢肌肉發(fā)育不良等多種組織病變。人的PAX3雜合突變可導(dǎo)致輕度的WS1表型，而復(fù)合雜合或純合PAX3突變則導(dǎo)致嚴(yán)重的WS3表型[62]。此外，PAX3突變還能引起顱面-耳聾-手綜合征(Craniofacial-deafness-hand syndrome, CDHS，MIM：122880)[63]。PAX3不僅在維持干細(xì)胞的多潛能性、細(xì)胞系遷移、分化增殖、凋亡和抑制終末分化中起重要作用[18]，而且在胚胎的神經(jīng)嵴及其衍生物的發(fā)育中起關(guān)鍵作用。Tremblay等[64]用轉(zhuǎn)基因神經(jīng)解剖標(biāo)記 PAX3在Splotch小鼠的神經(jīng)嵴發(fā)育中的作用，表明PAX3在神經(jīng)嵴源性結(jié)構(gòu)特別是顱神經(jīng)節(jié)和神經(jīng)的發(fā)育中起重要作用。神經(jīng)嵴細(xì)胞中PAX3的降低將導(dǎo)致神經(jīng)嵴細(xì)胞過早凋亡，引起相關(guān)發(fā)育疾病。Wu等[65]發(fā)現(xiàn)在神經(jīng)嵴中 PAX3的持續(xù)表達(dá)會(huì)導(dǎo)致小鼠出現(xiàn)腭裂和缺陷成骨，該研究為PAX3的下調(diào)表達(dá)在多能神經(jīng)嵴前體分化和顱腦發(fā)育過程中起到的重要作用提供了體內(nèi)證據(jù)。人和小鼠PAX基因表達(dá)水平下調(diào)影響神經(jīng)嵴的正常發(fā)育，說明神經(jīng)嵴的發(fā)育依賴 PAX3的正常功能，PAX3突變可導(dǎo)致多種神經(jīng)嵴源性細(xì)胞的功能異常。目前，PAX3調(diào)控神經(jīng)嵴發(fā)育的具體機(jī)制尚不明確，但在神經(jīng)嵴的發(fā)育過程中，PAX3通過調(diào)控編碼TYR受體的c-RET、信號(hào)分子Wnt1和轉(zhuǎn)化生長因子 β2(TGF-β2)等下游靶基因的表達(dá)促進(jìn)神經(jīng)嵴的遷移、分化和增殖[66～68]。c-RET、Wnt1和TGF-β2是神經(jīng)嵴發(fā)育的關(guān)鍵調(diào)控因子。c-RET酪氨酸激酶受體跨越神經(jīng)嵴源性細(xì)胞胞膜，是細(xì)胞的動(dòng)力生存和分化增殖所必需的。PAX3在神經(jīng)嵴源性細(xì)胞中通過調(diào)節(jié)c-RET的表達(dá)刺激腸道神經(jīng)節(jié)的正常遷移和發(fā)育[69]。PAX3直接與c-RET啟動(dòng)子上的增強(qiáng)子元件結(jié)合后協(xié)同SOX10激活c-RET表達(dá)，這種協(xié)同方式受染色質(zhì)乙?；{(diào)節(jié)。Wnt1是PAX3的下游基因，缺乏PAX3的小鼠胚胎背神經(jīng)管中的Wnt1表達(dá)減少[70]。神經(jīng)嵴細(xì)胞分化受PAX3和ZIC1的協(xié)同調(diào)控，Wnt可使神經(jīng)嵴細(xì)胞遷移前PAX3的表達(dá)增加，遷移后 PAX3表達(dá)下降，而 Wnt對(duì)神經(jīng)嵴細(xì)胞的這種作用可被骨形成蛋白4阻斷[71]。TGF-β2是調(diào)節(jié)細(xì)胞行為和影響細(xì)胞動(dòng)力、增殖分化和凋亡的關(guān)鍵因子。許多神經(jīng)嵴源性細(xì)胞十分依賴TGF-β2，研究表明TGF-β2純合突變小鼠可導(dǎo)致神經(jīng)嵴病，影響顱面骨結(jié)構(gòu)、耳、心臟和泌尿生殖系統(tǒng)。缺乏PAX3表達(dá)的突變小鼠胚胎與野生胚胎相比，TGF-β2表達(dá)明顯減少[72]。此外，在神經(jīng)嵴遷移早期，PAX3與FoxD3的比例還決定神經(jīng)嵴細(xì)胞向神經(jīng)/神經(jīng)膠質(zhì)細(xì)胞或是色素細(xì)胞轉(zhuǎn)變[73]。

性別決定區(qū)盒基因(SRY(sex determining region Y)-box 10, SOX10)位于染色體22q13.1，編碼的蛋白含有466個(gè)氨基酸，是高度保守的轉(zhuǎn)錄因子，由高活性組分結(jié)構(gòu)域(High mobility group, HMG)、Group E結(jié)構(gòu)域和C端轉(zhuǎn)錄激活域(TA)構(gòu)成，其中HMG域的主要功能是識(shí)別并結(jié)合DNA[74]。酵母雙雜交分析結(jié)果表明，SOX10通過HMG域的N端直接與KROX-20、PAX3、PAX6、HOXA3等至少17種可調(diào)節(jié)SOX10轉(zhuǎn)錄活性的蛋白相互作用[75]。SOX10為神經(jīng)嵴分化和少突細(xì)胞分化所必需，人的雜合SOX10突變會(huì)導(dǎo)致一些神經(jīng)嵴源性病變和腦白質(zhì)病變，相關(guān)疾病表型包括WS2和WS4[29]，外周和中樞髓鞘形成障礙，PCWH(OMIM：609136)[76]、HSCR(OMIM：142623)[77]和YDBS(OMIM：601706)[78]等。SOX10基因突變絕大部分為無義突變和移碼突變，導(dǎo)致終止密碼子提前出現(xiàn)并產(chǎn)生截短蛋白而致病[79]，此外，修飾基因的活性、突變導(dǎo)致的單倍體劑量不足以及其他原因引起的SOX10蛋白功能改變可部分解釋上述疾病表型的致病原因[80]。SOX10最先在NCC遷移早期的背神經(jīng)管中表達(dá)，然后隨著NCC逐漸分化而在周圍神經(jīng)系統(tǒng)、ENS和黑素細(xì)胞等衍生物如中表達(dá)。在人和成年小鼠中廣泛表達(dá)于大腦、心臟、肺、腎上腺、結(jié)腸、膀胱、胰腺、前列腺、睪丸、內(nèi)耳等各種組織器官中[81]。SOX10是神經(jīng)嵴源性細(xì)胞遷移分化的一種關(guān)鍵轉(zhuǎn)錄因子，可單獨(dú)或協(xié)同其他轉(zhuǎn)錄因子，通過與 MITF、DCT、TYR、TYRP1、RET、MPZ、GJB1和EDNRB等下游靶基因的啟動(dòng)子或增強(qiáng)子結(jié)合直接或間接參與黑色素合成。SOX10在黑素細(xì)胞和腸道神經(jīng)節(jié)的發(fā)育中發(fā)揮重要作用，可促進(jìn)胚胎神經(jīng)細(xì)胞和外周神經(jīng)系統(tǒng)的發(fā)育，不僅為胚胎發(fā)育早期所必需，而且對(duì)胚胎后期的小腸神經(jīng)系統(tǒng)的發(fā)育也非常重要[82]。而SOX10在內(nèi)耳發(fā)育早期廣泛表達(dá)[83,84]，提示SOX10突變導(dǎo)致的耳聾可能還有另外的機(jī)制。SOX10基因敲除小鼠模型的研究表明前體感覺祖細(xì)胞的表達(dá)減少會(huì)導(dǎo)致耳蝸?zhàn)兌蘙85]。這一觀察結(jié)果與SOX10突變耳聾患者內(nèi)耳MRI/CT掃描發(fā)現(xiàn)的形態(tài)學(xué)異常結(jié)果相同[85,86]。Mao等[87]利用神經(jīng)脊特異轉(zhuǎn)錄因子SOX10缺失小鼠模型來評(píng)估Schwann細(xì)胞缺陷在內(nèi)耳神經(jīng)支配中的作用，發(fā)現(xiàn)神經(jīng)脊來源的Schwann細(xì)胞為螺旋神經(jīng)節(jié)神經(jīng)元的遷移提供了終止信號(hào)，并能通過螺旋神經(jīng)節(jié)傳導(dǎo)促進(jìn)耳蝸Corti器的正確定位。

3.2 PAX3-SOX10-MITF通路參與色素異常表型相關(guān)的綜合征型耳聾的發(fā)病機(jī)制

PAX3-SOX10-MITF通路與色素異常相關(guān)耳聾綜合征的發(fā)病機(jī)制有重要聯(lián)系[88]。神經(jīng)嵴及其起源的組織細(xì)胞的發(fā)育過程復(fù)雜且受多個(gè)轉(zhuǎn)錄因子、信號(hào)通路和生長因子的調(diào)控[89]。WS致病基因MITF、PAX3、SOX10和SNAI2編碼的轉(zhuǎn)錄因子以及EDN3和EDNRB編碼的信號(hào)傳導(dǎo)分子都參與其中并發(fā)揮重要作用[90]。這些轉(zhuǎn)錄因子的調(diào)控作用與 WS等色素異常表型相關(guān)疾病的病理生理明顯相關(guān)，他們之間的相互作用以及他們與其他基因之間的相互作用構(gòu)成復(fù)雜的調(diào)控網(wǎng)絡(luò)調(diào)控 NCC源性細(xì)胞和組織尤其是黑素細(xì)胞的發(fā)育，表現(xiàn)為以 MITF為中心的調(diào)控與被調(diào)控的功能性聯(lián)系[91～93]。WS 致病基因 MITF、PAX3、SOX10之間存在相互作用。研究顯示SOX10與PAX3可單獨(dú)或協(xié)同激活并上調(diào) MITF 表達(dá)[94]。SOX10通過與MITF啟動(dòng)子的高度保守序列結(jié)合后使MITF轉(zhuǎn)錄活性提高 100倍，且 PAX3可增強(qiáng)這種作用。SOX10與 MITF協(xié)同激活負(fù)責(zé)黑色素合成的多巴色素互變異構(gòu)酶(Dopachrome tautomerase, DCT)，而PAX3拮抗MITF對(duì)DCT的轉(zhuǎn)錄[95]。同時(shí)MITF可調(diào)控SNAI2表達(dá)[28]，SOX10調(diào)控MITF對(duì)TYR表達(dá)的誘導(dǎo)和整個(gè)黑素細(xì)胞的分化[96]。SOX10在ENS和黑素細(xì)胞發(fā)育中都能夠調(diào)控EDNRB的表達(dá)[97]，而EDNRB又可通過信號(hào)傳導(dǎo)通路調(diào)控MITF轉(zhuǎn)錄[98](圖2，④⑤⑧PAX3-MITF-SOX10、MITF--EDNRB 通路)。本研究小組前期研究[56,57]表明，PAX3、SOX10與 MITF突變通過單倍體劑量不足分別導(dǎo)致WS1和WS2，突變蛋白可與其自身或正常的野生蛋白結(jié)合形成二聚體，使野生蛋白與靶基因 DNA不能正確結(jié)合而失去調(diào)節(jié)黑色素細(xì)胞靶基因的活性。此外，PAX3和SOX10基因突變可通過顯性負(fù)效應(yīng)抑制野生 PAX3和SOX10蛋白功能而導(dǎo)致嚴(yán)重的WS3和WS4。

除了 WS，PAX3-SOX10-MITF通路還可能與Leopard綜合征及Noonan綜合征等色素異常表型相關(guān)耳聾綜合征的發(fā)病機(jī)制有關(guān)。Leopard綜合征是一類以多發(fā)性黑斑為主要臨床表現(xiàn)的色素異常型綜合征，有15%～25%的患者出現(xiàn)感音神經(jīng)性耳聾，其最常見的致病基因是 PTPN11基因[99]。研究表明，PTPN11在 MET/Gab1復(fù)合物特殊信號(hào)中起重要作用，Gab1作為酪氨酸激酶受體c-MET的基質(zhì)，通過直接結(jié)合c-MET的結(jié)合域，參與c-MET的特異性分化。Gab1突變的過表達(dá)通過與PTPN11相互作用阻止HGF/SF誘導(dǎo)的MAPK通路的激活，從而在c-MET/Gab1的特殊信號(hào)中發(fā)揮重要作用[100](圖2，⑦ METPTPN11通路)。而肝細(xì)胞生長因子(HGF)/ c-MET的信號(hào)被認(rèn)為在黑素細(xì)胞的發(fā)展和黑色素瘤轉(zhuǎn)移中起關(guān)鍵作用。PAX3、SOX10以及MITF通過調(diào)節(jié)MET基因的表達(dá)在黑色素瘤細(xì)胞中起重要作用[101]；MITF通過上調(diào)MET的表達(dá)幫助肝細(xì)胞生長因子保護(hù)黑色素細(xì)胞并調(diào)控黑色素細(xì)胞瘤凋亡[102]。PAX3和MITF以及MET的啟動(dòng)子獨(dú)立結(jié)合，可以獨(dú)立調(diào)控并激活MET，而SOX10不能直接激活MET，但是可以通過協(xié)同 MITF和 PAX3激活 MET[101](圖 2，⑥ METMITF通路)。除了Leopard綜合征，PTPN11基因的突變還能在大約50%的Noonan綜合征病例中檢出。此外，RAF1基因的雜合突變也能同時(shí)在Leopard綜合征及Noonan綜合征患者中檢出[103]。

綜上所述，這些色素相關(guān)型耳聾綜合征除了共同具有色素發(fā)育異常和神經(jīng)性耳聾的臨床表型，還存在共同的致病基因。這些基因可能通過他們之間的相互作用以及他們與其他基因之間的相互作用構(gòu)成復(fù)雜的調(diào)控網(wǎng)絡(luò)調(diào)控 NCC 源性細(xì)胞和組織尤其是黑素細(xì)胞的發(fā)育，從而在伴有色素異常表型的綜合征型耳聾的發(fā)病機(jī)制中發(fā)揮作用。

3.3 以PAX3和SOX10為中心的基因互作通路參與小耳畸形表型相關(guān)綜合征的發(fā)病機(jī)制

SOX10和PAX3，以及通路中涉及的EYA1、SIX1、SALL1、HOX基因家族、EGR2(KROX-20)以及FGF3等基因與小耳畸形表型相關(guān)綜合征的發(fā)病機(jī)制有重要聯(lián)系。耳腮腎綜合征(Branchio-oto-renal syndrome，BOR)和Townes Brocks綜合征是小耳畸形相關(guān)綜合征里相對(duì)多見的兩個(gè)綜合征，二者都有混合型耳聾和小耳畸形的臨床表型[104,105]。EYA1和SALL1分別是這兩種綜合征的致病基因，與PAX家族和SIX1基因聯(lián)系密切[106,107]。在內(nèi)耳中，反式激活磷酸酶 EYA1與同源蛋白SIX1交互形成轉(zhuǎn)錄激活復(fù)合物，調(diào)節(jié)感應(yīng)和神經(jīng)細(xì)胞的增殖、存活以及內(nèi)耳發(fā)育過程中的分化誘導(dǎo)[108]。SALL1是EYA/SIX復(fù)合體的下游調(diào)控靶基因。SIX1直接結(jié)合SALL1的啟動(dòng)子并以劑量依賴性的方式誘導(dǎo)SALL1表達(dá)。而SIX1與PAX、EYA1基因及其產(chǎn)物在復(fù)雜的網(wǎng)絡(luò)中具有相互依存關(guān)系，形成正、負(fù)反饋回路，在腎臟發(fā)育過程中具有一定的網(wǎng)絡(luò)作用[109](圖 2，①②③ PAX-EYA-SIX-SALL1通路)。Fatemeh等[110]對(duì)來自伊朗的一個(gè)家系的小耳畸形患者的DNA序列進(jìn)行分析，結(jié)果發(fā)現(xiàn)一個(gè)HOXA2基因的同源結(jié)構(gòu)變異(p.Q186K)是該患者發(fā)生小耳畸形的重要原因。Hox基因家族編碼高度保守的轉(zhuǎn)錄因子，參與腮弓的發(fā)育。HOXA1基因失活導(dǎo)致小鼠外耳、中耳和內(nèi)耳的發(fā)育不良。而HOXA1/HOXB1復(fù)合物的突變則會(huì)導(dǎo)致無耳。而HOXA2可能作用于定義第二腮弓的特性以及耳廓形成的初始階段，在小鼠耳廓高表達(dá)。HOXB6和HOXA7基因缺失的小鼠表達(dá)出小耳畸形并伴有眼裂增寬，唇腭裂。Hox基因家族在各菱腦的特殊表達(dá)通過 EGR2的直接調(diào)節(jié)以控制后腦的分節(jié)[111,112](圖 2，⑩ EGR2-HOXA2通路)。SOX10作為神經(jīng)嵴細(xì)胞分化遷移中重要的轉(zhuǎn)錄因子之一，和EGR2在神經(jīng)嵴細(xì)胞遷移中共表達(dá)。在自激活過程中，SOX10是EGR2在神經(jīng)嵴細(xì)胞中自動(dòng)調(diào)節(jié)重要的共活化劑[113]。Reiprich等[113]研究表明，在雪旺氏細(xì)胞中，SOX10影響髓鞘形成，通過直接激活髓鞘基因和誘導(dǎo) EGR2調(diào)節(jié)外圍髓鞘的形成。SOX10與轉(zhuǎn)錄因子(POU3F1和POU3F2)協(xié)同作用激活EGR2后，再協(xié)同EGR2激活下游靶基因。這種依賴SOX10轉(zhuǎn)錄的連鎖模式在雪旺氏細(xì)胞和黑素細(xì)胞中一樣(圖 2，⑨ SOX10-EGR2通路)。伴有小耳畸形表型的 LAMM 綜合征目前已知的唯一致病基因是FGF3，研究表明LAMM綜合征與FGF3的隱性突變共分離[114]。FGF3與包括SOX10在內(nèi)的至少5個(gè)基因家族的調(diào)節(jié)基因直接或間接受到EGR2在神經(jīng)嵴細(xì)胞遷徙時(shí)r3/r5的控制，F(xiàn)GF3是后腦中控制EGR2表達(dá)的重要信號(hào)分子，抑制 FGF3的信號(hào)會(huì)下調(diào)EGR2的表達(dá)[115](圖2，?FGF3-EGR2通路)。

綜上所述，本文通過查閱大量文獻(xiàn)并結(jié)合本課題組前期的研究基礎(chǔ)，以SOX10和PAX3基因?yàn)橹行?，總結(jié)并繪制了與神經(jīng)嵴細(xì)胞的遷移和分化有關(guān)的基因相互作用網(wǎng)絡(luò)以及該通路涉及的多種綜合征型耳聾的關(guān)系圖(圖2)。

3.4 神經(jīng)嵴細(xì)胞遷移分化相關(guān)基因互作網(wǎng)絡(luò)涉及前庭導(dǎo)水管擴(kuò)大表型的致病機(jī)制

圖2 與神經(jīng)嵴細(xì)胞的遷移和分化有關(guān)的基因相互作用網(wǎng)絡(luò)及涉及的綜合征

值得注意的是，在上述與神經(jīng)嵴發(fā)育異常相關(guān)的綜合征型耳聾中，也存在不少合并有前庭導(dǎo)水管擴(kuò)大的內(nèi)耳畸形的報(bào)道。Madden等[116]通過對(duì) 9個(gè)Warrdenburg綜合征患兒和正常兒童進(jìn)行統(tǒng)計(jì)學(xué)分析比較，發(fā)現(xiàn)所有患者均有聽力損失，50%的患兒出現(xiàn)前庭導(dǎo)水管中段擴(kuò)大。Elmaleh-Bergès等[117]對(duì)收集到的由SOX10導(dǎo)致的Waardenburg綜合征患者進(jìn)行內(nèi)耳影像學(xué)觀察，發(fā)現(xiàn)雙側(cè)內(nèi)耳異常(包括半規(guī)管的缺如和發(fā)育不全，前庭導(dǎo)水管擴(kuò)大以及耳蝸畸形)極有可能與Waardburg綜合征的致病基因SOX10有關(guān)。耳腮腎綜合征患者也經(jīng)常伴有前庭導(dǎo)水管擴(kuò)大表型。Stinckens等[118]對(duì)一個(gè)耳腮腎綜合征大家系進(jìn)行了研究，結(jié)果顯示部分患者伴有擴(kuò)大的前庭導(dǎo)水管和漸進(jìn)性感音神經(jīng)性耳聾。Kemperman等[119]對(duì)兩名耳腮腎綜合征患者(父子)進(jìn)行長期聽力隨訪檢測以及巖骨 CT成像研究，發(fā)現(xiàn)進(jìn)行波動(dòng)性聽力損失和熱量功能減退與前庭導(dǎo)水管擴(kuò)大相關(guān)。Propst等[120]通過 CT斷層掃描將耳腮腎綜合征的患者和正常人群進(jìn)行比較，結(jié)果發(fā)現(xiàn)，耳腮腎綜合征患者內(nèi)耳CT表現(xiàn)和對(duì)照組之前存在明顯差異(30/39，76.9%)，最常見的特征性表現(xiàn)有：耳蝸頂圈發(fā)育不良、面神經(jīng)偏向耳蝸內(nèi)側(cè)、漏斗狀內(nèi)聽道、咽鼓管異常開放。鑒于前庭導(dǎo)水管擴(kuò)大在耳腮腎綜合征中出現(xiàn)較多，Ito等[121]以 SIX1基因突變的耳腮腎綜合征的患者為研究對(duì)象，探尋SIX1基因突變和前庭導(dǎo)水管擴(kuò)大之前的關(guān)系；發(fā)現(xiàn)在SIX1的Y129C突變可能導(dǎo)致前庭導(dǎo)水管擴(kuò)大和耳腮腎綜合征的發(fā)生。以上研究提示：與神經(jīng)嵴細(xì)胞的遷移和分化有關(guān)的基因相互作用網(wǎng)絡(luò)可能在 WS等色素異常以及小耳畸形相關(guān)的耳聾綜合征的發(fā)病機(jī)制中起重要作用，還可能參與前庭導(dǎo)水管擴(kuò)大表型的致病機(jī)制。

4 結(jié)語與展望

以往耳聾研究往往局限于對(duì)單個(gè)基因突變致病機(jī)理進(jìn)行研究。而如今有許多臨床表型無已法用單基因突變進(jìn)行解釋。在機(jī)體內(nèi)，基因編碼產(chǎn)物往往不是單獨(dú)發(fā)揮作用，而是形成較大的復(fù)合體，在特定時(shí)間和空間行使特定的功能，所以基因功能的研究應(yīng)突破單基因研究模式，主張研究基因間的相互作用，最終在一個(gè)復(fù)雜的基因網(wǎng)絡(luò)中全面認(rèn)識(shí)基因功能。綜合征型耳聾基因涉及多個(gè)系統(tǒng)，卻可以導(dǎo)致很多共同的臨床表型，這表明它們之間可能存在著潛在的共同通路和相互作用網(wǎng)絡(luò)。其中某一環(huán)節(jié)出現(xiàn)功能障礙即有可能影響網(wǎng)絡(luò)的整體功能。這方面的研究有過成功的例子，Usher綜合征中多種USH1蛋白(Cadherin23、Harmnin、Protocadherin和Myosin VIIa)之間存在著復(fù)雜的相互作用關(guān)系已經(jīng)得到證實(shí)。Brodbeck等[111]的研究提出理論，由于進(jìn)化是保守的，基因相互作用的網(wǎng)絡(luò)在不同物種的不同器官中可能存在共同的作用機(jī)制和通路。以PAXEYA-SIX調(diào)節(jié)通路為例，小鼠的遺傳和生化研究表明，SIX1與PAX、EYA1在腎臟發(fā)育過程中有一定的網(wǎng)絡(luò)作用，這些基因和它們各自的產(chǎn)物作用在復(fù)雜的網(wǎng)絡(luò)中具有相互依存關(guān)系，形成正負(fù)反饋回路。鑒于在果蠅中也有同樣的階梯效應(yīng)，推斷PAX-EYA-SIX調(diào)節(jié)通路廣泛存在于哺乳動(dòng)物腎臟發(fā)育過程中。這種相互作用關(guān)系在果蠅和小鼠中有同樣的效應(yīng)，那么在耳發(fā)育過程中也可能存在相似的網(wǎng)絡(luò)效應(yīng)。目前國內(nèi)外的綜合征型耳聾相關(guān)研究分散，規(guī)模小，人群樣本量小且病例資源有限，鑒定的基因種類和數(shù)量嚴(yán)重不足，而且即便是已鑒定的致病基因?qū)ζ渲旅@機(jī)制的研究亦不系統(tǒng)和深入，導(dǎo)致尚有絕大部分綜合征型耳聾患者不能得到明確的基因診斷和臨床治療。Waardenburg綜合征、先天性小耳畸形相關(guān)綜合征以及前庭導(dǎo)水管擴(kuò)大表型等SHL的發(fā)病機(jī)制存在共性，都與神經(jīng)嵴細(xì)胞遷移分化相關(guān)的基因互作網(wǎng)絡(luò)有關(guān)，未來針對(duì)綜合征型耳聾潛在的共同通路和致病機(jī)制進(jìn)行系統(tǒng)地研究，可為綜合征型耳聾的研究提供新的思路和理論基礎(chǔ)。

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