陳曉麗 郭 金 鄒繼珍 盧曉琳 包怡華 吳麗華 牛 勃

神經(jīng)管畸形(neural tube defects,NTDs)在世界范圍內(nèi)的發(fā)病率約為1‰[2,3]。中國NTDs發(fā)病率高達(dá)27.4‰，每年有8～10萬例NTDs患兒出生，占先天畸形的20%～25%[4]。本課題組前期研究發(fā)現(xiàn)NTDs在山西省的發(fā)病率高達(dá)199.38‰[5]。

NTDs為多因素復(fù)雜疾病，是遺傳和環(huán)境因素共同作用的結(jié)果[6～9]。既往研究發(fā)現(xiàn)母體營養(yǎng)代謝的異常，特別是母體葉酸缺乏為NTDs的主要危險(xiǎn)因素[10,11]，而孕期補(bǔ)充葉酸可以減少50%～70%的NTDs發(fā)生[12～14]。葉酸在體內(nèi)的主要功能是參與DNA合成及提供甲基供體。在孕期給予低葉酸飲食喂養(yǎng)的大鼠，其子代出現(xiàn)DNA總體低甲基化[15]，而給葉酸缺乏的大鼠補(bǔ)充葉酸可以逆轉(zhuǎn)DNA總體甲基化水平的降低[16]?；蚪MDNA總體甲基化水平影響基因組穩(wěn)定性，而特定基因啟動(dòng)子區(qū)的甲基化水平影響該基因轉(zhuǎn)錄[17]?；蚪M不穩(wěn)定引發(fā)染色體重組性疾病以及腫瘤發(fā)生[18～20]，因特定基因組或特定基因的異常甲基化引起的出生缺陷有Rett綜合征、Prader-Willi/Angelman綜合征以及Beckwith-Wiedemann綜合征[21]。

DNA修復(fù)通路在控制基因突變和維持基因組穩(wěn)定等方面具有重要作用，與一些神經(jīng)系統(tǒng)遺傳病發(fā)生有關(guān)[22,23]。錯(cuò)配修復(fù)基因(mismatch repair gene，MMR)是細(xì)胞內(nèi)負(fù)責(zé)對(duì)堿基錯(cuò)配進(jìn)行修復(fù)的基因，現(xiàn)有研究提示DNA修復(fù)通路基因可能在神經(jīng)管早期發(fā)育中發(fā)揮作用，如BRCA1基因敲除的小鼠出現(xiàn)脊柱裂和無腦兒表型[24]，NTDs患兒腦組織中MMR蛋白表達(dá)水平顯著下降[25]。

本課題組前期研究結(jié)果已經(jīng)驗(yàn)證母體血清低葉酸水平是NTDs的危險(xiǎn)因素[26]。基于此，本研究以醫(yī)院為基礎(chǔ)進(jìn)行病例-對(duì)照研究，通過比較NTDs組與對(duì)照組DNA總體甲基化和7個(gè)MMR啟動(dòng)子區(qū)的甲基化水平，了解NTDs胎兒是否存在DNA甲基化修飾改變，為母體葉酸相對(duì)不足導(dǎo)致NTDs胎兒出現(xiàn)DNA甲基化修飾異常的假設(shè)尋找依據(jù)。

1 方法

1.1 研究對(duì)象 所有標(biāo)本均來源于山西省呂梁地區(qū)縣級(jí)醫(yī)院及婦幼保健院引產(chǎn)的胎兒，由首都兒科研究所病理科醫(yī)生完成解剖診斷。NTDs組：B超診斷為NTDs而進(jìn)行引產(chǎn)的胚胎，經(jīng)解剖證實(shí)有1種或多種形式的NTDs畸形；對(duì)照組：同期非病理性引產(chǎn)的胚胎，經(jīng)解剖證實(shí)不存在任何先天畸形和胎兒發(fā)育遲緩。兩組引產(chǎn)胚胎分別取腦組織和皮膚組織各25 mg，迅速液氮凍存。Array-CGH芯片檢測(cè)排除大片段微染色體異常(>1 Mb)。以孕周和孕婦年齡對(duì)兩組進(jìn)行匹配。

1.2 倫理和知情同意 該研究經(jīng)首都兒科研究所倫理委員會(huì)批準(zhǔn)，入組胚胎標(biāo)本均經(jīng)家屬的知情同意用于研究。

1.3 甲基化水平檢測(cè) 采用Qiagen組織/血樣DNA提取試劑盒(DNeasy Blood & Tissue kit)提取腦組織和皮膚組織的DNA，A260計(jì)算DNA濃度。用DNA甲基化檢測(cè)試劑盒(Epigentek，F(xiàn)armingdale,NY)完成DNA總體甲基化檢測(cè)。

采用MS-MLPA試劑盒(ME011-A1,MRC-Holland，Amsterdam，荷蘭)對(duì)腦組織MMR啟動(dòng)子區(qū)的甲基化水平進(jìn)行分析，共包括7個(gè)MMR(MLH1，MSH2，MSH6，MSH3，MLH3，PMS2和MGMT)。每個(gè)基因檢測(cè)3～4個(gè)HhaⅠ酶切位點(diǎn)，共24個(gè)目的位點(diǎn)(3個(gè)PMS2探針，6個(gè)MLH1探針，4個(gè)MSH2探針，3個(gè)MSH3探針，3個(gè)MSH6探針，3個(gè)MGMT探針，2個(gè)MLH3探針)，同時(shí)有8個(gè)內(nèi)參位點(diǎn)(內(nèi)參位點(diǎn)內(nèi)沒有HhaⅠ酶切位點(diǎn))。每個(gè)標(biāo)本分成兩管，一管進(jìn)行HhaⅠ酶切消化(HhaⅠ+)，一管不進(jìn)行HhaⅠ酶切消化(HhaⅠ-)，利用ABI3730(諾賽基因有限公司，北京)對(duì)PCR進(jìn)行峰值測(cè)定，然后用Genemarker 1.95軟件(SoftGenetics,State College,PA)對(duì)基因甲基化數(shù)據(jù)進(jìn)行圖像采集和甲基化分析。如待測(cè)DNA的GCGC位點(diǎn)未甲基化，則探針DNA復(fù)合物被HhaⅠ酶切開，致使PCR片段擴(kuò)增失敗，反之則可擴(kuò)增出PCR片段。以(消化后目的位點(diǎn)峰值/消化前目的位點(diǎn)峰值)/(消化后內(nèi)參位點(diǎn)峰值/消化前內(nèi)參位點(diǎn)峰值) 計(jì)算每個(gè)樣本的甲基化水平。

2 結(jié)果

2.1 一般情況 2006年4月至 2007年12月共收集到符合匹配條件的65例NTDs胚胎和48例正常對(duì)照胚胎。兩組孕婦的年齡和孕周分布見表1，其構(gòu)成比無顯著差異。

2.2 腦組織和皮膚組織的DNA總體甲基化水平 首先兩組腦組織和皮膚組織的DNA總體甲基化水平數(shù)據(jù)進(jìn)行正態(tài)性檢驗(yàn)，數(shù)據(jù)呈正態(tài)分布。由圖1可見，DNA總體甲基化在腦組織和皮膚組織顯示為不同的分布。皮膚組織的DNA總體甲基化水平顯著高于腦組織。進(jìn)一步比較NTDs組和對(duì)照組DNA甲基化水平，發(fā)現(xiàn)兩組腦組織DNA總體甲基化水平存在顯著差異，NTDs組顯著低于對(duì)照組(5.3%vs6.5%，P<0.001)，而兩組皮膚組織DNA總體甲基化水平差異無統(tǒng)計(jì)學(xué)意義(13.3%vs13.1%,P>0.05)。

表1 NTDs組和對(duì)照組的一般情況

Tab 1 Basic information of NTD-affected cases and controls

NTDs[n(%)]Controls[n(%)]P1)Women'sage/y≤2539(60.9)19(39.6)0.074-3017(26.6)18(37.5)≥318(12.5)11(22.9)GA/weeks≤2026(40)27(56.3)0.224-2419(29.2)11(22.9)≥2520(30.8)10(20.8)

Notes 1)Pvalue fromχ2test; y: year; GA: gestational age

圖1 腦組織及皮膚組織DNA總體甲基化水平在NTDs組和對(duì)照組的比較

Fig 1 Comparison of global DNA methylation levels of brain and skin tissue in NTD-affected cases and controls

Notes 1) Global DNA methylation levels of brain in NTD-affected cases were lower than those in controls,P<0.001

Logistical 回歸分析結(jié)果顯示，腦組織DNA總體低甲基化水平(<對(duì)照組P25)使NTDs發(fā)生的危險(xiǎn)提高4.98倍(表2)。

表2 DNA總體甲基化水平與NTDs發(fā)生危險(xiǎn)度的Logistic回歸結(jié)果

Tab 2 Logistic regression of global DNA methylation and formation of NTDs

Level1)Controls[n(%)]NTDs[n(%)]OR(95%CI)AdjustedOR2)(95%CI)P3)Low(P75)12(25.0)6(9.2)1(reference)1(reference)

Notes GA: gestational age. 1) Global DNA methylation levels in cases were categorized into three subgroups according to the percentiles of controls; 2)Model was adjusted for women′s age and gestational week. 3)Pvalue fromχ2test

2.3 MMR啟動(dòng)子區(qū)的甲基化水平 NTDs組和對(duì)照組各38例胚胎腦組織進(jìn)行了MMR啟動(dòng)子區(qū)甲基化水平檢測(cè)，MS-MPLA結(jié)果如圖2所示。

圖2 待測(cè)樣本消化前后MS-MLPA結(jié)果

Fig 2 The MS-MLPA results for test sample before(A) and after(B) HhaⅠdigestion

Notes A showed the genomic copy number of 8 reference loci(blue dots) and 24 target loci(green dots). Green line represented the threshold of genomic copy number(0.75-1.25). In this case,the genomic copy number of 24 target loci was normal compared with reference loci. B showed the methylation ratio of 8 reference loci(blue dots) and 24 target loci(red and green dots). Green line represented the threshold of methyletion ratio(30%). The reference loci(blue dots) had high methylation ratio because they had no HhaⅠsite. 4 target loci(MSH2-246,MLH1-148,MLH3-202,MSH3-283) had high methyaltion above the threshold because they were resistant to HhaⅠdigestion,other target loci(green dots) had low methyaltion ratio under the threshold because they were digested by HhaⅠ

如表3所示，在7個(gè)MMR基因的24個(gè)位點(diǎn)上，有3個(gè)目的位點(diǎn)為高甲基化狀態(tài)(MSH2-246，MLH1-148，MLH3-202，甲基化百分比>80%)，20個(gè)目的位點(diǎn)為低甲基化狀態(tài)(甲基化百分比<20%)，MSH3-283為中等甲基化狀態(tài)(20%～80%)。通過比較NTDs組和對(duì)照組24個(gè)目的位點(diǎn)甲基化百分比均值，發(fā)現(xiàn)MSH6和PMS2啟動(dòng)子區(qū)上3個(gè)位點(diǎn)存在顯著差異(MSH6-301：2.5%vs3.7%，P<0.01；PMS2-328：5.7%vs6.7%,P<0.05；PMS2-142：2.0%vs2.7%,P<0.05)。另有MGMT啟動(dòng)子區(qū)1個(gè)位點(diǎn)(MGMT-193)的甲基化水平差異接近顯著水平(P=0.05)。

MMRlociControls(n=38)NTDs(n=38)PMSH2-1841.1±0.41.1±0.20.555MSH2-4002.1±1.12.4±0.80.335MSH2-2720.000.00NAMSH2-24692.4±10.789.7±5.00.493MSH6-3012.5±1.43.7±1.20.0051)MSH6-2090.10±0.180.000.068MSH6-1601.4±0.51.5±0.50.639MLH1-2371.4±0.71.7±0.80.159MLH1-14899.5±9.997.3±9.70.545MLH1-2650.000.00NAMLH1-1980.000.00NAMLH1-1660.9±0.40.9±0.20.704MLH1-2920.5±0.50.6±0.50.377MSH3-2208.4±1.78.2±1.50.744MSH3-3462.2±0.82.4±0.60.522MSH3-28345.3±1.252.4±1.30.119PMS2-3285.7±1.36.7±0.90.011PMS2-1422.0±0.72.7±1.20.028PMS2-1541.3±2.00.5±0.50.431MGMT-37326.2±5.425.9±5.40.887MGMT-1934.1±1.53.3±1.10.050MGMT-3191.5±0.61.6±0.60.719MLH3-3550.000.19NAMLH3-20290.3±42.9100NA

Notes NA: not available for student′sttest; 1)Pvalue remained significant after Bonferroni correction at 0.005

3 討論

DNA甲基化修飾對(duì)于胚胎的發(fā)育至關(guān)重要[27]。在受精后的6～8 h，胚胎經(jīng)歷了廣泛的去甲基化過程，此時(shí)DNA的甲基化程度會(huì)成比例降低。在植入期，胚胎又發(fā)生了快速的重新甲基化過程[28]。既往研究發(fā)現(xiàn)，阻斷Dnmt3b的表達(dá)可以干擾神經(jīng)管的閉合[29]。在對(duì)雞胚的研究中發(fā)現(xiàn)，當(dāng)雞胚暴露在甲基化抑制劑中，其神經(jīng)系統(tǒng)的發(fā)育會(huì)出現(xiàn)延遲，并且這種延遲與所暴露的劑量成正比[30]。這些研究結(jié)果均提示NTDs形成過程中可能出現(xiàn)DNA甲基化修飾異常。

本研究采用病例-對(duì)照的方法，發(fā)現(xiàn)NTDs組胚胎腦組織DNA總體甲基化水平降低，增加了患NTDs的風(fēng)險(xiǎn)，推測(cè)腦組織DNA的低甲基化狀態(tài)可能是母體葉酸缺乏導(dǎo)致胎兒發(fā)生NTDs的潛在致病機(jī)制之一。

DNA總體甲基化分布存在組織特異性，唐韶青等[31]運(yùn)用MSAP技術(shù)對(duì)豬、牛、羊、小鼠、雞和鴨不同組織的DNA總體甲基化水平做了分析，發(fā)現(xiàn)同一種動(dòng)物不同組織的DNA總體甲基化水平存在差異。本研究發(fā)現(xiàn)NTDs組和對(duì)照組的DNA總體甲基化水平差異僅表現(xiàn)在腦組織，而兩組皮膚組織的DNA總體甲基化水平無顯著差異，說明DNA總體甲基化模式在胎兒時(shí)期就出現(xiàn)了組織特異性。

在胚胎發(fā)育早期，一些神經(jīng)發(fā)育關(guān)鍵基因的啟動(dòng)子區(qū)域的甲基化也需要重新修飾。本課題組前期研究應(yīng)用MS-MLPA(MRC-Holland,ME002-A1)分析了MGMT、MSH6啟動(dòng)子區(qū)甲基化異常和NTDs的關(guān)系，發(fā)現(xiàn)NTDs組(特別是腦部NTDs組)的MSH6(1250-L0798)、MGMT(5670-L5146)位點(diǎn)甲基化狀態(tài)顯著低于對(duì)照組(3.5%vs5.3%，16.3%vs27.2%)[32]。本研究結(jié)果顯示，MGMT-193的差異水平接近顯著(P=0.05)，進(jìn)而比較了2個(gè)研究中的探針設(shè)計(jì)位置，發(fā)現(xiàn)MGMT-193和MGMT(5670-L5146)為MGMT基因的同一GCGC位點(diǎn)，再次證實(shí)MGMT基因啟動(dòng)子異常甲基化與NTDs的關(guān)系，而檢驗(yàn)P值不同可能源于2個(gè)研究中腦部脊柱裂在NTDs組所占比例不同。本研究還發(fā)現(xiàn)NTDs組MSH6-301位點(diǎn)甲基化狀態(tài)顯著高于對(duì)照組，與本課題組的前期結(jié)果不同。通過分析探針位置，發(fā)現(xiàn)MSH6-301、MSH6-209、MSH6-160位點(diǎn)與MSH6(1250-L0798)所處基因組位置不同，說明了基因啟動(dòng)子CpG區(qū)甲基化修飾的復(fù)雜性，即使同一基因的同一區(qū)域，各位點(diǎn)的甲基化狀態(tài)也存在差異。Liu等[33]研究NTDs胎兒的MLH1、MSH2啟動(dòng)子甲基化水平，發(fā)現(xiàn)NTDs組和正常對(duì)照組并無顯著差異，且各位點(diǎn)的甲基化狀態(tài)也不一致，如MSH2均為低甲基化水平位點(diǎn)，而MLH1有2個(gè)高甲基化水平位點(diǎn)。本研究分析了4個(gè)MSH2位點(diǎn)和6個(gè)MLH1位點(diǎn)，雖然研究位點(diǎn)不同，但也證實(shí)僅MSH2-246和MLH1-148為高甲基化水平位點(diǎn)，其余位點(diǎn)都處于低甲基化水平，并且NTDs組和對(duì)照組甲基化水平無顯著差異。除已報(bào)道的MSH2、MSH6和MGMT外，本研究還新發(fā)現(xiàn)1個(gè)MMR(PMS2)啟動(dòng)子甲基化水平在NTDs組和對(duì)照組存在顯著差異，提示了PMS2啟動(dòng)子甲基化異常和NTDs的關(guān)系。上述結(jié)果深化了對(duì)神經(jīng)管發(fā)育過程中MMR的DNA甲基化修飾的認(rèn)識(shí)。

總之，本研究對(duì)NTDs胚胎腦組織DNA總體甲基化、MMR啟動(dòng)子區(qū)甲基化水平進(jìn)行了分析，發(fā)現(xiàn)NTDs胚胎腦組織DNA總體甲基化水平顯著低于對(duì)照組，DNA總體低甲基化水平顯著增加了NTDs患病風(fēng)險(xiǎn)。同時(shí)發(fā)現(xiàn)NTDs組的MSH6、PMS2基因啟動(dòng)子區(qū)甲基化水平不同于對(duì)照組。這些結(jié)果為進(jìn)一步研究神經(jīng)管發(fā)育過程中DNA總體低甲基化的發(fā)生發(fā)展、MMR甲基化修飾奠定基礎(chǔ)，也為明確葉酸缺乏與DNA甲基化異常、NTDs發(fā)生的關(guān)系，葉酸預(yù)防NTDs機(jī)制提供理論依據(jù)。

目前關(guān)于基因特定位點(diǎn)甲基化檢測(cè)方法有MS-MLPA、甲基化特異性PCR(MS-PCR)、結(jié)合重亞硫酸鹽的限制性內(nèi)切酶法(COBRA)、甲基化特異探針雜交法。MS-MLPA技術(shù)將甲基化敏感性限制性內(nèi)切酶HhaⅠ和探針雜交技術(shù)結(jié)合，通過分析模板DNA酶切后的擴(kuò)增比例來定量甲基化程度。該技術(shù)缺點(diǎn)是所分析位點(diǎn)受限制性內(nèi)切酶位點(diǎn)(GCGC序列)限制，因而在本研究中，對(duì)于MMR的啟動(dòng)子區(qū)非HhaⅠ位點(diǎn)，其甲基化程度無法依賴MS-MLPA進(jìn)行定量，今后需要利用其他甲基化研究技術(shù)繼續(xù)分析MMR的啟動(dòng)子區(qū)甲基化水平。

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