饒書權(quán), 杜廷福, 許琪

中國醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所, 北京 100005

外顯子組測(cè)序在人類疾病中的應(yīng)用

饒書權(quán), 杜廷福, 許琪

中國醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所, 北京 100005

據(jù)估計(jì), 約 85%的人類遺傳變異集中在蛋白編碼區(qū), 因此對(duì)全部的蛋白編碼區(qū)(外顯子組)進(jìn)行重測(cè)序,可以快速、有效地鑒定人類疾病遺傳變異。以往鑒定孟德爾遺傳病的致病基因多采用連鎖分析結(jié)合候選定位克隆的方法, 不僅耗時(shí)長, 而且成功率低。2009年, 科學(xué)家第一次應(yīng)用外顯子組測(cè)序在4名弗里曼謝爾登綜合征(常染色體顯性遺傳病)中發(fā)現(xiàn)了位于MYH3中的點(diǎn)突變, 顯示出外顯子組測(cè)序在孟德爾遺傳病致病基因鑒定中的強(qiáng)大功效。就復(fù)雜疾病而言, 傳統(tǒng)的關(guān)聯(lián)研究, 包括全基因組關(guān)聯(lián)研究(GWAS), 雖然鑒定了大量的常見變異,但對(duì)低頻變異和罕見變異的檢測(cè)能力十分有限; 深度測(cè)序的發(fā)展為解決上述問題提供了良好的契機(jī)。文章就外顯子組測(cè)序在人類疾病中的應(yīng)用進(jìn)行了綜述。

外顯子組測(cè)序; 孟德爾遺傳疾病; 復(fù)雜疾病

醫(yī)學(xué)研究的一個(gè)重要領(lǐng)域是探索人類疾病的遺傳變異, 從而為疾病的診斷、預(yù)防及其治療提供理論基礎(chǔ)[1]。遺傳學(xué)理論和技術(shù)的進(jìn)步, 極大地豐富了遺傳學(xué)知識(shí), 越來越多的遺傳變異被陸續(xù)鑒定出來。近幾十年來, 連鎖分析(Linkage analysis)和關(guān)聯(lián)研究(Association study), 包括候選基因關(guān)聯(lián)研究(Candidate gene association study)和全基因組關(guān)聯(lián)研究(Genome-wide association study, GWAS)分別在鑒定孟德爾遺傳病的致病基因及復(fù)雜疾病的易感基因中發(fā)揮了重要作用。連鎖分析的基本思想為：根據(jù)疾病在家系中特有的遺傳模式, 利用遺傳標(biāo)記, 如短串聯(lián)重復(fù)序列(Short tandem repeats, STR)和單核苷酸多態(tài)性(Single nucleotide polymorphism, SNP),將致病基因定位在染色體上的某一小段區(qū)域; 然后利用測(cè)序技術(shù)來尋找該區(qū)域內(nèi)的致病變異。通過連鎖分析, 已成功鑒定約 1/3的孟德爾遺傳病的致病基因[2]。然而, 對(duì)于家系成員過少(或不齊全)、致病基因外顯率過低或位點(diǎn)異質(zhì)性高的孟德爾遺傳病,連鎖分析的效能則會(huì)明顯減弱[3]。

迄今, 通過候選基因關(guān)聯(lián)研究和全基因組關(guān)聯(lián)研究, 科研人員已鑒定出眾多復(fù)雜疾病, 包括精神分裂癥[4,5]、腫瘤[6]等的易感位點(diǎn)。然而, 所有已經(jīng)揭示的易感位點(diǎn)只能解釋疾病的一小部分遺傳度[7,8]。此外, 隨著易感位點(diǎn)的最小等位基因頻率(Minor allele frequency, MAF)逐漸下降, 關(guān)聯(lián)研究達(dá)到足夠檢驗(yàn)效能所需的樣本含量往往呈指數(shù)級(jí)增長[8,9], 因此通過關(guān)聯(lián)研究鑒定得到的位點(diǎn)多為致病效力微弱的常見變異(MAF＞0.05), 而對(duì)低頻變異(MAF＜0.05)和罕見變異(MAF＜0.01)的檢測(cè)能力十分有限[8]。

自2005年以來, 二代測(cè)序技術(shù)得到了極大的改善, 測(cè)序成本大幅度降低。研究人員逐漸開始通過對(duì)人類基因組進(jìn)行重測(cè)序來尋找疾病的易感基因[10]。人類基因組大約含180 000個(gè)外顯子, 總長約30 Mb,該區(qū)域包含蛋白質(zhì)編碼合成所需要的絕大部分信息,被稱為外顯子組(Whole exome), 大約85%的致病突變位于外顯子區(qū)域[11]?；蚪M靶向捕獲技術(shù)的發(fā)展使得研究人員可以高效、特異地對(duì)外顯子組進(jìn)行測(cè)序[12,13]。2009年8月, Ng等[14]對(duì)4名無親緣關(guān)系的弗里曼謝爾登綜合征患者(已知該病的致病基因?yàn)镸YH3)及8名正常對(duì)照的DNA樣本進(jìn)行外顯子組測(cè)序, 通過對(duì) 12個(gè)樣本的測(cè)序數(shù)據(jù)進(jìn)行比較分析, 準(zhǔn)確找出了位于 MYH3中的突變, 這是外顯子組測(cè)序技術(shù)第一次成功應(yīng)用于疾病致病基因的鑒定, 并預(yù)示了其作為遺傳學(xué)研究工具的廣闊前景。

本文就全外顯子組測(cè)序在孟德爾疾病和復(fù)雜疾病中的應(yīng)用進(jìn)行綜述。

1 外顯子組測(cè)序的基本原理

外顯子組測(cè)序指利用特殊的手段對(duì)全外顯子組進(jìn)行富集, 并進(jìn)行高通量測(cè)序的技術(shù)方法。其基本流程包括外顯子區(qū)域序列的富集、高通量測(cè)序及測(cè)序數(shù)據(jù)的生物信息學(xué)分析。

測(cè)序平臺(tái)以Illumina公司的Solexa和Hiseq二代測(cè)序技術(shù)為主[15,16]。近兩年來, LifeTechnologies推出了基于多重PCR的AmpliSeq外顯子捕獲方法, 該捕獲方法結(jié)合 Ion torrent代測(cè)序平臺(tái)因能顯著縮短實(shí)驗(yàn)周期而逐漸受到研究人員的親睞[17,18]。隨著測(cè)序技術(shù)的快速發(fā)展, 市場(chǎng)上還涌現(xiàn)出以 PacBio的SMRT技術(shù)和Oxford的Nanopore技術(shù)為代表的三代測(cè)序技術(shù)[19,20], 極大地改善了測(cè)序準(zhǔn)確度。

生物信息學(xué)分析的目的是挖掘變異位點(diǎn), 包括SNP和 Indels。首先是通過質(zhì)控排除測(cè)序過程中低質(zhì)量的Reads, 然后將高質(zhì)量的Reads與參考基因組進(jìn)行比對(duì), 統(tǒng)計(jì)SNP和Indels, 并對(duì)這些變異位點(diǎn)進(jìn)行注釋。Qian等[21]開發(fā)了一套標(biāo)準(zhǔn)的數(shù)據(jù)處理流程,可以高效快速的自動(dòng)化處理高通量測(cè)序數(shù)據(jù), 得到變異位點(diǎn)列表。研究人員再根據(jù)疾病的種類、測(cè)序樣本的數(shù)量等設(shè)計(jì)方案對(duì)這些變異位點(diǎn)進(jìn)行深入分析, 最終確定候選變異位點(diǎn)。

2 外顯子組測(cè)序在孟德爾遺傳病中的應(yīng)用

孟德爾遺傳病, 又叫單基因病, 是指由于單個(gè)基因突變而導(dǎo)致的疾病, 它常以孟德爾遺傳模式存在于家系中。2010年, Ng等[22]利用外顯子組測(cè)序成功地鑒定了米勒綜合征的致病基因 DHODH; 之后,外顯子組測(cè)序技術(shù)被廣泛應(yīng)用于鑒定各種孟德爾遺傳疾病致病突變。截止2013年1月, 已有超過150種孟德爾遺傳病的致病基因通過外顯子組測(cè)序被鑒定出來[23,24], 如Ohdo綜合征(KAT6B)[25]、陣發(fā)性運(yùn)動(dòng)障礙(PRRT2)[26]、Bohring-Opitz綜合征(ASXL1)[27]、脊椎干骺端發(fā)育不良(KIF22)[28]、高酯血癥(APOE)[29]、先天性靜止性夜盲(LRIT3)[30]。

對(duì)于孟德爾遺傳病而言, 疾病的遺傳模式在很大程度上影響了實(shí)驗(yàn)設(shè)計(jì)和數(shù)據(jù)分析, 充分利用家系信息能夠顯著縮小候選致病基因的范圍[24]。孟德爾遺傳病的遺傳模式可以大致分為隱性遺傳和顯性遺傳兩大類。

2.1 隱性遺傳病

隱性遺傳病包括非近親結(jié)婚隱性遺傳病、近親結(jié)婚隱性遺傳病以及X-連鎖隱性遺傳病。

非近親結(jié)婚隱性遺傳病的致病突變一般為復(fù)合雜合突變(Compound heterozygous mutations), 即患者的雙親所攜帶的突變位點(diǎn)不一致, 而單個(gè)突變并不致病, 只有當(dāng)個(gè)體同時(shí)攜帶兩個(gè)突變位點(diǎn)時(shí)才患病(圖1A)。致病突變鑒定策略為：首先保留患者中具有兩個(gè)突變的基因, 若某個(gè)基因的兩個(gè)突變分別存在于正常雙親中, 則該基因有可能是該病的致病基因。Janneke等[31]通過對(duì)兩個(gè)符合非近親結(jié)婚隱性遺傳模式的癲癇性腦病家系進(jìn)行外顯子組測(cè)序, 成功找出致病基因DOCK7。另一方面, 雖然非近親結(jié)婚隱性遺傳病的致病基因?yàn)榧兒屯蛔兊那闆r較少,但在分析的時(shí)候不能完全排除這種可能性, 如 John等[32]通過對(duì)一個(gè)非近親結(jié)婚隱性遺傳模式的神經(jīng)元蠟樣脂褐質(zhì)沉積癥家系進(jìn)行分析, 發(fā)現(xiàn)一個(gè)純和致病基因突變KCTD7。近親結(jié)婚的隱性遺傳病的致病基因一般為純和突變(Homozygous mutations)(圖1B)。致病突變鑒定策略為：若某一突變?cè)诨颊咧袨榧兒?而在正常雙親中為雜合, 那么該突變有可能為該病的致病突變。Christiano等[33]對(duì)一近親結(jié)婚的多毛癥家系進(jìn)行外顯子組測(cè)序, 確定一個(gè)致病基因ABCA5;該基因其在患者中為純和突變, 而父母中則為該基因雜合突變的攜帶者?？傊? 由于個(gè)體攜帶的復(fù)合雜合突變或純和突變數(shù)目稀少, 因此鑒定具有以上兩類隱性遺傳模式的孟德爾遺傳病的致病基因相對(duì)容易。

施索仁認(rèn)為，中國市場(chǎng)對(duì)馬士基來說非常重要，貿(mào)易訂單的減少將對(duì)馬士基的物流業(yè)務(wù)產(chǎn)生不利影響。馬士基集團(tuán)主要是在集裝箱運(yùn)輸、物流、碼頭運(yùn)營、石油和天然氣開采與生產(chǎn)，以及與航運(yùn)和零售行業(yè)相關(guān)其他活動(dòng)中，為客戶提供服務(wù)，馬士基集團(tuán)旗下的馬士基航運(yùn)是全球最大的集裝箱承運(yùn)輸公司。

X-連鎖隱性遺傳病的致病基因位于X染色體上(圖1C)。若某一突變存在于男性患者的X染色體上,且在女性攜帶者中為雜合, 那么該突變有可能為疾病的致病突變。符合這類遺傳模式的疾病, 致病基因的鑒定同樣比較簡(jiǎn)單。但值得注意的是, 在家系結(jié)構(gòu)不完善的情況下, 研究人員常常難以區(qū)分 X-連鎖隱性遺傳模式和常染色體隱性遺傳模式。Diamond-Blackfan貧血是一個(gè)典型的例子：研究人員首先根據(jù)常染色體隱性遺傳模式對(duì)該家系進(jìn)行分析, 沒能找到致病基因; 但當(dāng)研究人員采用 X-連鎖隱性遺傳模式進(jìn)行分析時(shí), 成功發(fā)現(xiàn)了致病基因GATA1, 該基因在另外一組人群中得到了驗(yàn)證[34]。對(duì)家系遺傳模式的準(zhǔn)確判斷, 可大大節(jié)省致病基因篩選的時(shí)間和成本, 如 Ginevra等[35]通過對(duì)一個(gè)具有兩例小腦性共濟(jì)失調(diào)患者的三代家系進(jìn)行分析,發(fā)現(xiàn)該家系符合X-連鎖隱性遺傳模式, 便針對(duì)X染色體外顯子組進(jìn)行測(cè)序, 成功地找到了其致病基因PMCA3。

2.2 顯性遺傳病

利用外顯子組測(cè)序鑒定顯性遺傳疾病致病基因則相對(duì)比較困難(圖 1D), 這是因?yàn)橥ㄟ^測(cè)序得到的候選基因數(shù)目龐大, 而致病突變的強(qiáng)致病性通常導(dǎo)致家系過小, 使得對(duì)相應(yīng)致病基因的鑒定達(dá)不到足夠的檢驗(yàn)效能[24]。其分析策略為：尋找患者共有的雜合突變, 排除正常人中存在的雜合突變, 剩下的突變則可能為疾病的致病突變。

對(duì)于符合顯性遺傳模式的遺傳病, 其致病基因的分析很多時(shí)候需要結(jié)合連鎖分析等方法。Liu等[36]利用連鎖分析(全基因組微衛(wèi)星標(biāo)記)將兩個(gè)家族發(fā)作性疼痛病家系的致病基因定位在3p22.3-p21.32上,然后利用外顯子組測(cè)序在兩個(gè)家系中發(fā)現(xiàn)了SCN11A基因(3p22.2)的兩個(gè)錯(cuò)義突變, 最后結(jié)合家系內(nèi)共分離分析以及 SCN11A基因功能研究, 確定SCN11A為家族發(fā)作性疼痛一個(gè)新的致病基因。

圖1 孟德爾隱性遺傳和顯性遺傳模式示意圖

3 外顯子組測(cè)序在復(fù)雜疾病中的應(yīng)用

復(fù)雜疾病, 如腫瘤、精神疾病、心血管疾病等,其典型特征是風(fēng)險(xiǎn)因素多樣, 遺傳異質(zhì)性高, 給研究人員對(duì)致病基因的鑒定帶來極大的困難。

截止2014年5月, 研究人員利用GWAS鑒定出5804個(gè)具有強(qiáng)關(guān)聯(lián)信號(hào)的SNP(p＜ 1x 10-8)[37]。但所有這些常見變異都是微效基因, 并且只能解釋一小部分遺傳度[38,39]。目前普遍接受的觀點(diǎn)是利用GWAS篩查得到的SNP只是作為一個(gè)標(biāo)簽, 而與該標(biāo)簽處于連鎖不平衡的罕見變異才是真正的致病變異[40]。

外顯子組測(cè)序用于復(fù)雜疾病的遺傳研究, 其策略主要有以下兩種：基于人群的外顯子組測(cè)序, 致力于尋找頻發(fā)突變(Recurrent mutations)或新生突變(De novo mutations); 基于家系的外顯子組測(cè)序, 致力于尋找遺傳變異(Inherited mutations)。研究人員認(rèn)為, 對(duì)于復(fù)雜疾病而言, 散發(fā)病例的易感基因多來源于de novo突變, 而家系的易感基因則多來自于遺傳突變[41]。

3.1 惡性腫瘤

突變過度積累可導(dǎo)致惡性腫瘤的發(fā)生。腫瘤細(xì)胞高度的遺傳異質(zhì)性極大地制約了對(duì)腫瘤遺傳因素的發(fā)掘。此外, 腫瘤突變大部分為新生體細(xì)胞突變,傳統(tǒng)的 GWAS在腫瘤遺傳學(xué)研究中越來越捉襟見肘。Chang等[42]利用外顯子組測(cè)序?qū)?種常用的癌癥細(xì)胞系進(jìn)行測(cè)序分析, 結(jié)果顯示對(duì)眾多已知突變位點(diǎn)的分型結(jié)果與用Affymetrix SNP array 6.0芯片的分型結(jié)果具有高度的一致性(95%)。這表明外顯子組測(cè)序應(yīng)用于癌癥基因組的研究不僅廉價(jià), 而且十分可靠。近年來, 利用全外顯子組測(cè)序已經(jīng)成功鑒定出多種腫瘤的眾多變異。Brastianos等[43]利用外顯子組測(cè)序發(fā)現(xiàn)在 92%(11/12)的顱咽管瘤患者中發(fā)現(xiàn)CTNNB1具有突變：其中3例乳頭狀顱咽管瘤患者為位于 BRAF編碼區(qū)的點(diǎn)突變(p.Val600Glu), 擴(kuò)大樣本對(duì)該位點(diǎn)進(jìn)行分型發(fā)現(xiàn), 約 95%(36/39)的患者具有該點(diǎn)突變。Li等[44]在57例膽囊癌-癌旁組織對(duì)照樣本中利用外顯子組測(cè)序發(fā)現(xiàn)多個(gè)患者中存在TP53(47.1%)、ERBB3(11.8%)和KRAS(7.8%)的突變;此外, ErbB通路相關(guān)基因在 36.8%的患者中存在突變, 提示該通路可能廣泛參與膽囊癌的發(fā)生發(fā)展。Kakiuchi等[45]對(duì)30例彌漫性胃癌樣本進(jìn)行外顯子組測(cè)序, 并在57例樣本中進(jìn)行驗(yàn)證發(fā)現(xiàn)25.3%的彌漫性胃癌患者中存在RHOA的點(diǎn)突變, 這些點(diǎn)突變主要有Tyr42、Arg5和Gly17等。

3.2 重性精神疾病

重性精神疾病包括精神分裂癥、孤獨(dú)癥、智力發(fā)育障礙、重性抑郁癥以及雙相情感障礙等。研究表明, de novo突變?cè)谥匦跃窦膊≈邪缪葜种匾慕巧?。Xu等[46]利用外顯子組測(cè)序?qū)?3名精神分裂癥患者以及22名正常對(duì)照的de novo突變進(jìn)行分析, 發(fā)現(xiàn) 51%(27/53)的精神分裂癥患者攜帶至少一個(gè)de novo突變, 而在正常人中該比例約為32%。Xu等[47]在另一篇有關(guān)精神分裂癥 de novo 拷貝數(shù)變異(CNV)的分析中, 發(fā)現(xiàn)約有 10%(15/152)的精神分裂癥患者攜帶de novo CNVs, 而該比例在正常人中僅為1.3%(2/159)。兩項(xiàng)研究表明超過60%的精神分裂癥患者攜帶 de novo突變。Takata等[48]在 231例精神分裂癥患者中發(fā)現(xiàn) 2個(gè)位于 SETD1A的 de novo突變, 該基因編碼一個(gè)組蛋白甲基轉(zhuǎn)移酶, 可以調(diào)節(jié)染色質(zhì)的結(jié)構(gòu)。

除精神分裂癥之外, 研究人員在其他精神疾病,如孤獨(dú)癥、智力發(fā)育障礙中都發(fā)現(xiàn)了大量 de novo突變。Gregor等[49]通過對(duì)智力障礙核心家系(triofamily)進(jìn)行測(cè)序分析, 在CTCF中發(fā)現(xiàn)一個(gè)de novo錯(cuò)義突變以及一個(gè)de novo移碼突變。CTCF基因編碼染色質(zhì)結(jié)構(gòu)重塑蛋白, 位于CTCF中的de novo可以通過影響染色質(zhì)的結(jié)構(gòu)進(jìn)而影響增強(qiáng)子與啟動(dòng)子之間的相互作用。Tavassoli等[50]在孤獨(dú)癥患者中發(fā)現(xiàn)位于基因SCN2A的點(diǎn)突變(c.476 + 1G ＞ A), 該突變可能導(dǎo)致編碼的蛋白質(zhì)縮短并介導(dǎo)mRNA降解。

在鑒定得到大量de novo突變的同時(shí), 我們也應(yīng)該注意到, 并不是找到的de novo突變都是疾病的真正易感基因, 因?yàn)槿魏蝹€(gè)體都有一定的機(jī)會(huì)攜帶 de novo突變。如何設(shè)計(jì)實(shí)驗(yàn)并采用新的研究策略是遺傳學(xué)家需要重點(diǎn)思考的問題。

此外, 大量重性精神疾病患者也存在家族聚集傾向, 這提示重性精神疾病在特定條件下也可能符合孟德爾遺傳模式。遺傳學(xué)家按照孟德爾遺傳病的分析模式, 已成功鑒定出多個(gè)精神疾病致病基因,解釋了部分精神疾病的遺傳基礎(chǔ)。例如, 研究人員對(duì)多個(gè)患有神經(jīng)系統(tǒng)疾病的家系進(jìn)行外顯子組測(cè)序,通過分析發(fā)現(xiàn)這些患者具有共同的突變基因 CLP1,該基因負(fù)責(zé)調(diào)控細(xì)胞中的 tRNA代謝, 攜帶該基因突變的兒童表現(xiàn)大腦畸形、智力障礙、癲癇、感知和運(yùn)動(dòng)缺陷等癥狀[51,52]。

3.3 心血管疾病

心血管疾病是老年人死亡的重要原因。根據(jù)遺傳因素的不同, 可以將心血管疾病分為兩類, 即單基因型(包括離子通道病、家族型血脂異常等)和多基因型(如心肌肥厚、擴(kuò)張型心肌病等)。先天性心臟病是一類病因不明的心血管疾病。Glessner等[53]通過拷貝數(shù)芯片分析和外顯子組測(cè)序發(fā)現(xiàn), 與正常對(duì)照相比, 先天性心臟病患者攜帶更多的拷貝數(shù)變異(CNV); 結(jié)合外顯子組測(cè)序的數(shù)據(jù), 作者發(fā)現(xiàn) ETS1和CTBP2可能是先天性心臟病新的易感基因。Theis等[54]利用外顯子組測(cè)序?qū)σ粋€(gè)符合隱性遺傳模式的擴(kuò)張型心肌病進(jìn)行分析, 發(fā)現(xiàn)了位于GATAD1中的一個(gè)點(diǎn)突變。此外, 外顯子組測(cè)序還成功在家族型血脂過少(ANGPTL3)[55]、重型血脂過多(ABCG)[56]以及家族型心肌擴(kuò)張(BAG3)等疾病中發(fā)現(xiàn)了新的易感基因。

利用外顯子組測(cè)序挖掘心臟、肺和血液的遺傳變異, 美國國家心臟、肺和血液研究所(NHLBI)還發(fā)起了 Grand Opportunity Exome Sequencing Project (ESP)。該計(jì)劃共收集了超過200 000人的外顯子組測(cè)序數(shù)據(jù), 取得了廣泛的成果[57,58]。

2013年10月, Yang等[59]對(duì)外顯子組測(cè)序的實(shí)驗(yàn)方法、生物信息分析等流程進(jìn)行了詳細(xì)介紹, 并提示外顯子組測(cè)序可用于遺傳病的臨床診斷。隨后, Nature和The New England Journal of Medicine雜志分別發(fā)布了人類疾病遺傳變異研究指南和高通量測(cè)序臨床應(yīng)用指南, 對(duì)高通量測(cè)序?qū)嶒?yàn)設(shè)計(jì)、數(shù)據(jù)分析、功能驗(yàn)證、臨床應(yīng)用等提供了指導(dǎo)準(zhǔn)則, 加深了人們對(duì)外顯子組測(cè)序在人類疾病中應(yīng)用的認(rèn)識(shí)[60,61]。

4 外顯子組測(cè)序的技術(shù)限制

盡管外顯子組測(cè)序在人類疾病的遺傳學(xué)研究中取得了極大的成功, 但該技術(shù)本身也存在一些缺點(diǎn),主要包括外顯子捕獲效率及測(cè)序正確率。

為了系統(tǒng)評(píng)價(jià)外顯子組測(cè)序技術(shù)對(duì)致病突變的檢測(cè)效率, Gilissen等[62]對(duì) 51個(gè)外顯子組測(cè)序數(shù)據(jù)的 37 424個(gè) SNP位點(diǎn)(Human Genome Mutation Database)進(jìn)行了分析。共有35 296個(gè)突變位點(diǎn)被成功捕獲, 捕獲成功率為94.3%; 測(cè)序深度在10x以上的突變位點(diǎn)約占全部位點(diǎn)的 80.8%, 其中不能被成功檢測(cè)的位點(diǎn)所在區(qū)域大都顯示過高或過低的 GC含量[63]。研究表明, 對(duì) GC豐富的基因組區(qū)域而言,打斷捕獲和基于多重PCR的捕獲方式均具有較高的捕獲能力; 但對(duì) AT豐度的基因組區(qū)域而言, AmpliSeq在捕獲時(shí)會(huì)出現(xiàn)很大的偏差[64]。除基因組本身過高或過低的GC或AT含量及其特殊結(jié)構(gòu)之外,所有外顯子組捕獲試劑盒的探針設(shè)計(jì)都不能實(shí)現(xiàn)對(duì)外顯子區(qū)域100%的覆蓋度, 使得部分外顯子區(qū)域成為測(cè)序盲區(qū)。隨著全基因組測(cè)序費(fèi)用的進(jìn)一步降低,采用全基因組測(cè)序, 替代外顯子組測(cè)序, 進(jìn)行遺傳變異的挖掘?qū)?huì)是必然的趨勢(shì)。

測(cè)序深度對(duì)突變位點(diǎn)的檢測(cè)能力有較大影響。Hoischen等[65]利用芯片捕獲結(jié)合深度測(cè)序的方法對(duì) 5名常染色體隱性遺傳共濟(jì)失調(diào)患者的已知致病突變進(jìn)行再驗(yàn)證, 結(jié)果只發(fā)現(xiàn)6/7的已知突變。將測(cè)序數(shù)據(jù)量提高 3倍以后, 研究人員成功檢測(cè)到第 7個(gè)突變。此外, 不管是哪種測(cè)序平臺(tái), 測(cè)序過程中都不可避免地存在一些錯(cuò)誤[66,67]。研究人員發(fā)現(xiàn)與Hiseq測(cè)序平臺(tái)相比, 在相同測(cè)序深度的條件下, Ion Torrent可以檢測(cè)出更多的變異位點(diǎn), 但其假陽性也相應(yīng)提高[68]。Robasky等[69]總結(jié)了高通量測(cè)序?qū)嶒?yàn)中產(chǎn)生的錯(cuò)誤主要有 3大類, 分別來自樣品制備、文庫制備以及測(cè)序和成像過程。在測(cè)序過程中, 從樣品制備到數(shù)據(jù)分析, 都有可能產(chǎn)生錯(cuò)誤, 而其中一些錯(cuò)誤是可以避免的。

5 展 望

隨著新一代測(cè)序技術(shù)的快速發(fā)展, 全基因組測(cè)序和全外顯子組測(cè)序的應(yīng)用越來越廣泛[70], 由于與疾病相關(guān)的大部分功能性變異基本集中在染色體的外顯子區(qū), 通過外顯子組測(cè)序能夠迅速獲得外顯子區(qū)域的遺傳信息。與全基因組測(cè)序相比, 外顯子組測(cè)序成本更低、覆蓋度更廣、冗余數(shù)據(jù)更少, 可以快速有效地發(fā)掘疾病的致病基因或易感基因。但是任何一個(gè)外顯子組捕獲平臺(tái)的捕獲效率都不能達(dá)到100%, 使得部分外顯子組序列不能被捕獲, 而且越來越多的研究顯示, 非編碼區(qū)在疾病的發(fā)生發(fā)展中也同樣扮演著十分重要的角色。因此, 如果僅僅對(duì)外顯子組進(jìn)行測(cè)序, 可能會(huì)丟失部分重要的遺傳信息。隨著高通量技術(shù)的發(fā)展, 測(cè)序成本越來越低, 目前已經(jīng)實(shí)現(xiàn)1000美元全基因組測(cè)序, 相比之下, 全基因組測(cè)序在未來的研究中可能具有更大的發(fā)展空間。

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(責(zé)任編委: 胡松年)

The application of exome sequencing in human diseases

Shuquan Rao, Tingfu Du, Qi Xu

Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China

It is estimated that approximately 85% of human disease mutations are located in protein coding regions, and therefore selectively sequencing all protein coding regions (exome) would be cost-effective and an alternative strategy to identify variants of disease. Linkage analysis followed by candidate positional cloning has been used for identifying disease-causing genes of Mendelian disorders for a long time; however, this approach showed not only time-consuming but also low success rate. In 2009, scientists successfully identified one missense mutation in MYH3 among four individuals with Freeman Sheldon syndrome (one autosomal dominant disease) through exome sequencing, suggesting that exome sequencing could be a powerful tool for the identification of Mendelian disease variants. As for complex diseases, though traditional association studies and genome-wide association studies (GWAS) have identified a large number of common variants, their application in identification of low-frequency or rare variants isquite limited. The development of the next-generation sequencing technology provides us an opportunity to deal with the problem. In this review, we summarize the application of exome sequencing in human diseases.

exome sequencing; Mendelian disorder; complex disease

2014-07-30;

2014-10-13

國家自然科學(xué)基金項(xiàng)目(編號(hào)：31222031), 中央高?；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)資金項(xiàng)目(編號(hào)：2012S05)和協(xié)和青年科研基金項(xiàng)目(編號(hào)：2012J09)資助

饒書權(quán), 博士研究生, 研究方向：醫(yī)學(xué)遺傳學(xué)。E-mail: raoshuquantongji@163.com杜廷福, 博士研究生, 研究方向：醫(yī)學(xué)遺傳學(xué)。E-mail:dutingfu2912@163.com饒書權(quán)和杜廷福同為第一作者。

許琪, 博士, 研究員, 研究方向：醫(yī)學(xué)遺傳學(xué)。E-mail: xuqi@pumc.edu.cn

10.3724/SP.J.1005.2014.1077

時(shí)間: 2014-10-16 16:05

URL: http://www.cnki.net/kcms/detail/11.1913.R.20141016.1605.004.html