陳曉麗 郭 金 王 珺 王立文 丁秀原 張 霆 吳柏林

兒童發(fā)育遲緩(developmental delay,DD)和智力低下(mental retardation,MR)發(fā)病率約為3%[1]。盡管傳統(tǒng)細(xì)胞遺傳學(xué)檢測(cè)(常規(guī)染色體G帶分析)、FISH和新近發(fā)展的多重連接依賴探針擴(kuò)增法(multiplex ligation-dependent probe amplification, MLPA)等技術(shù)可提高M(jìn)R/DD患兒的病因檢出率，但仍有50%的患兒病因不明[2]。近年來(lái)，隨著微陣列技術(shù)分辨率的提高，國(guó)外科研人員利用微陣列比較基因組雜交(array-comparative genomic hybridization, Array-CGH)技術(shù)對(duì)不明原因MR/DD患兒開(kāi)展了全基因組拷貝數(shù)變異(copy number variations, CNVs)的研究，發(fā)現(xiàn)部分MR/DD患兒存在罕見(jiàn)CNVs，從而識(shí)別出一系列新的微缺失或重復(fù)綜合征[3,4]。中國(guó)至今尚未見(jiàn)這方面的研究報(bào)道。中國(guó)兒童MR/DD患病基數(shù)大，是神經(jīng)科主要就診人群，其中至少有2/3病因不明[5]。本研究應(yīng)用高分辨Array-CGH技術(shù)，對(duì)不明原因MR/DD患兒進(jìn)行了初步的全基因組CNVs篩查，了解可能與MR/DD相關(guān)的罕見(jiàn)基因組CNVs在中國(guó)人群不明原因MR/DD患兒中的檢出率，以此評(píng)估Array-CGH對(duì)不明原因MR/DD可能的遺傳病因診斷作用。

1 方法

1.1 研究對(duì)象

1.1.1 MR的診斷標(biāo)準(zhǔn) 根據(jù)不同的年齡段，分別采用《兒心0～4歲精神發(fā)育量表》和《中國(guó)韋氏智力量表》測(cè)定兒童的發(fā)育商(DQ)或智商( IQ )，同時(shí)運(yùn)用《嬰兒-初中生社會(huì)適應(yīng)性能力量表》進(jìn)行社會(huì)適應(yīng)性能力評(píng)價(jià)。IQ或DQ<70，同時(shí)伴社會(huì)適應(yīng)性能力低下者診斷為MR；>5歲稱為MR,<5歲稱為DD；以下統(tǒng)稱為MR/DD。

1.1.2 研究對(duì)象篩選標(biāo)準(zhǔn) 不明原因的MR/DD患兒指排除出生時(shí)產(chǎn)傷、生后中樞神經(jīng)系統(tǒng)感染和(或)頭顱損傷、染色體G帶檢查異常和(或)其他已知遺傳性綜合征和遺傳病，同時(shí)符合以下至少1個(gè)條件：①伴發(fā)有其他體表和(或)內(nèi)臟畸形，如先天性心臟病、脊柱畸形、多或少指(趾)等；②母親具有異常妊娠史，包括多次流產(chǎn)、胎死宮內(nèi)、死產(chǎn)或出生缺陷；③患兒診斷為孤獨(dú)癥或具有孤獨(dú)癥譜系障礙(autism spectrum disorder, ASD)；④患兒具有典型面部特征或異常面容；⑤患兒存在嚴(yán)重的生長(zhǎng)落后或發(fā)育遲緩。

1.1.3 研究對(duì)象剔除標(biāo)準(zhǔn) 所有患兒完成簡(jiǎn)單尿液篩查以排除遺傳代謝性疾病，針對(duì)某些疑似綜合征患兒，如脆性X染色體綜合征(fragile X syndrome)或 Rett綜合征, 采用Southern Blotting或PCR、MLPA進(jìn)行排除檢查。

1.1.4 倫理審核 所有患兒均為臨床病例，患兒監(jiān)護(hù)人填寫書面知情同意書或口頭同意參加全基因組CNVs檢測(cè)。本研究獲得首都兒科研究所倫理委員會(huì)批準(zhǔn)。

1.2 Array-CGH檢測(cè)方法 按照美國(guó)波士頓兒童醫(yī)院臨床分子診斷實(shí)驗(yàn)室的標(biāo)準(zhǔn)方法進(jìn)行操作[6]。QIAGEN 抽提外周抗凝血的基因組DNA，獲得吸光度值和濃度后，取3～6 μg DNA于50 μL體系，37℃消化2 h(Alu Ⅰ和Rsa Ⅰ聯(lián)合消化)； QIAprep Spin Miniprep Kit進(jìn)行DNA純化，重新測(cè)定吸光度值后，取2 500 ng DNA, 用BioPrime labeling kit進(jìn)行熒光標(biāo)記(cy5標(biāo)記對(duì)照標(biāo)本，cy3標(biāo)記MR/DD標(biāo)本)，37℃ 2 h后終止反應(yīng)；再次MicroCon YM-30 純化熒光標(biāo)記DNA后，將對(duì)照DNA和患兒DNA等量混合后加入雜交體系，94℃變性，37℃ 0.5 h，上樣到Oligo 244 K芯片上，65℃雜交爐孵育72 h后先用洗脫液1洗脫5 min，再用洗脫液2洗脫1 min， 迅速進(jìn)行微陣列掃描，采用Feature Extraction 9.0進(jìn)行數(shù)據(jù)提取至DNA analystic 5.0軟件進(jìn)行CNVs分析。以上儀器和試劑均有商品化供應(yīng)(Invitrogen和Agilent公司)。男女性對(duì)照DNA樣本購(gòu)自Invitrogen公司。

針對(duì)所發(fā)現(xiàn)的罕見(jiàn)CNVs，盡可能采用雙親樣本重復(fù)Array-CGH檢測(cè)，了解其是否為新生(De novo)CNV。

1.3 MR/DD相關(guān)CNVs 的評(píng)估方法 針對(duì)所有發(fā)現(xiàn)的CNVs，首先將這些CNVs與國(guó)際基因組CNVs多態(tài)性數(shù)據(jù)庫(kù)(database of genomic variants,DGV)和UCSC brower(build 18)比對(duì)，了解其是否為罕見(jiàn)CNVs(rare CNVs)或常見(jiàn)CNVs(common CNVs)，然后根據(jù)美國(guó)波士頓兒童醫(yī)院遺傳診斷實(shí)驗(yàn)室的CNVs 評(píng)估分析標(biāo)準(zhǔn)將CNVs分為6類(圖1)：①常見(jiàn)CNVs：至少有2例以上片段大小幾乎完全相同的CNVs或者80%以上區(qū)域完全重疊的相似CNVs，曾在DGV和UCSC brower中有報(bào)道；②罕見(jiàn)CNVs，且目前已明確與MR/DD相關(guān)(已發(fā)現(xiàn)的微缺失/重復(fù)綜合征)；③罕見(jiàn)CNVs,但很可能與MR/DD相關(guān)， 曾被DECIPHER數(shù)據(jù)庫(kù)、美國(guó)波士頓兒童醫(yī)院遺傳診斷實(shí)驗(yàn)室的aCGH-CNVs 數(shù)據(jù)庫(kù)報(bào)道，或被既往的MR/DD微陣列基因組研究文獻(xiàn)所報(bào)道，且已報(bào)道的CNVs大小幾乎完全相同或者至少80%區(qū)域重疊；④新生CNVs：在正常父母樣本中未曾發(fā)現(xiàn)，該 CNVs雖然在研究文獻(xiàn)中未曾報(bào)道，但含有與MR/DD可能相關(guān)的重要基因；⑤臨床意義不明確的CNVs：該CNVs未曾報(bào)道，且父母未能參與研究；⑥家族性CNVs：已在正常父母樣本中證實(shí)。 以上分類②～④均屬于與MR/DD相關(guān)或很有可能相關(guān)的CNVs，對(duì)于此類CNVs，該區(qū)域內(nèi)的基因名稱來(lái)自UCSC brower(build 18)。

圖1 CNVs 評(píng)估分析平臺(tái)

Fig 1 The workflow for CNVs evaluation

Notes MR:mental retardation;DD:developmental delay

1.4 文獻(xiàn)比對(duì)分析 在PubMed數(shù)據(jù)中，采用“unknown” or “unexplained”、“mental retardation” or “developmental delay”和“array”作為檢索詞，并組合成不同的檢索式進(jìn)行檢索。時(shí)間為2006年1月1日至2009年7月31日，檢索到一系列利用微陣列技術(shù)對(duì)不明原因MR/DD人群進(jìn)行全基因組CNVs研究的文獻(xiàn)，比對(duì)分析以確認(rèn)本研究所發(fā)現(xiàn)的CNVs 在既往研究中是否有報(bào)道。涵蓋的研究方法不局限于Oligo CGH array，還包括BAC-CGH array和SNP array。

2 結(jié)果

2.1 研究對(duì)象的一般情況 2004年7月至2008年7月在首都兒科研究所共收集不明原因MR/DD患兒111例，均為漢族，平均年齡為6歲(1個(gè)月至16歲)，男女比例為1.775(71∶40), 86例(77.4%)伴有生長(zhǎng)落后或發(fā)育遲緩；29例(26.1%)伴有其他畸形和(或)神經(jīng)系統(tǒng)異常，包括先天性心臟病，多指或趾，骨骼或脊柱異常，驚厥，刻板或孤獨(dú)樣癥狀；13例(11.7%)患兒的母親具有異常妊娠史；32例(28.8%)患兒具有MR/DD家族史；45例(40.5%)患兒有典型面部特征。37/111例患兒完成了常規(guī)染色體G帶檢查，均提示正常染色體核型。

2.2 Array-CGH的結(jié)果及數(shù)據(jù)比對(duì)分析 28/111例患兒中發(fā)現(xiàn)36個(gè)CNVs(重復(fù)16個(gè)，缺失20個(gè))，發(fā)生概率為32.4%(36個(gè)/111例)，CNVs平均長(zhǎng)度為1 326 kb(29～8 760 kb)(表1)。

4/28例患兒獲得父母血樣進(jìn)行親本鑒定，以了解患兒的CNVs是否為家族性遺傳。發(fā)現(xiàn)2例患兒具有新生CNVs；另2例為父親遺傳，其中1例遺傳自智力或神經(jīng)、精神發(fā)育缺陷的父親，另1例父親的智力或神經(jīng)、精神發(fā)育情況不詳。

對(duì)28例患兒所發(fā)現(xiàn)的36個(gè)罕見(jiàn)CNVs,利用美國(guó)波士頓兒童醫(yī)院遺傳診斷實(shí)驗(yàn)室的CNVs評(píng)估分析平臺(tái)，結(jié)合DECIPHER數(shù)據(jù)庫(kù)和既往MR/DD相關(guān)的Oligo CGH array、BAC-CGH array和SNP array的研究報(bào)告，發(fā)現(xiàn)22個(gè)CNVs可能與MR/DD相關(guān)。由于部分患兒同時(shí)攜帶MR/DD相關(guān)CNVs和未報(bào)道CNVs，故最終評(píng)估確認(rèn)19例患兒具有MR/DD相關(guān)CNVs，1例患兒的CNVs臨床意義不明確(表1)。因此，攜帶MR/DD致病相關(guān)CNVs的患兒檢出率為17.1%(19/111例)。

19個(gè)與MR/DD相關(guān)的罕見(jiàn)CNVs中，1例為16p11.2微缺失綜合征，Array-CGH結(jié)果見(jiàn)圖2；2例為Pelizaeus-Merzbacher??；2例涉及15q11-13的Prader-Willi綜合征/Angelman綜合征(PWS/AS)核心區(qū)域，Array-CGH結(jié)果見(jiàn)圖3；1例為Potocki-Lupski綜合征，Array-CGH結(jié)果見(jiàn)圖4；1例為Williams-Beuren綜合征；1例為16p13.11微缺失綜合征。

表1 不明原因MR/DD 患兒中CNVs類型特點(diǎn)

Notes 1) 16p11.2 Microdeletion syndrome; 2) Prader-Willi/Angelman syndromes (PWS/AS); 3) Williams-Beuren syndrome; 4) Atypical PWS; 5) Pelizaeus-Merzbacher disease;6) Potacki-Lupski syndrome; 7) 16p13.11 microdeletion syndrome; 8) father was NA, not maternal; NA: not available;-:not related

圖2 16p11.2微缺失綜合征患兒的Array-CGH 結(jié)果圖

Fig 2 Array-CGH data of a patient with 16p11.2 deletion

Notes A showed a 606 kb deletion in 16p11.2 region from patient 185. Each probe was represented as a single dot and plots on X axis according to its genome position. Duplication/deletion was shown respectively as red/green dot and normal as black one. The green/red bar was represented for the affected region. B showed the distribution of Refseq gene in UCSC Browser (build 18) and known common CNVs in DGV in enlarged affected region. The bottom red bar represents for known common CNVs and upper scattered blue bar was represented for Refseq gene

圖3 PWS患兒的Array-CGH 結(jié)果圖

Fig 3 Array-CGH data of a patient with 15q11.2-13 deletion associated with PWS

Notes A showed an approximately 5 Mb deletion at 15q11.2-13 from patient 232. Each probe was represented as a single dot and plots on X axis according to its genome position. Duplication/deletion was shown respectively as red/green dot and normal as black one. The green/red bar was represented for the affected region. B showed the distribution of Refseq gene in UCSC Browser (build 18) and known common CNVs in DGV in enlarged affected region. The bottom red bar was represented for known common CNVs and upper scattered blue bar was represented for Refseq gene

圖4 Potocki-Lupski綜合征患兒的Array-CGH 結(jié)果圖

Fig 4 Array-CGH data of a patient with Potocki-Lupski syndrome

Notes A showed an approximately 5 Mb duplication in 17p12-11.2 region from patient 221. Each probe was represented as a single dot and plots on X axis according to its genome position. Duplication/deletion was shown respectively as red/green dot and normal as black one. The green/red bar was represented for the affected region. B showed the distribution of Refseq gene in UCSC Browser (build 18) and known common CNVs in DGV in enlarged affected region. The bottom red bar was represented for known common CNVs and upper scattered blue bar was represented for Refseq gene

2.3 文獻(xiàn)比對(duì)分析 在PubMed數(shù)據(jù)庫(kù)通過(guò)文獻(xiàn)檢索，共獲得26篇涉及MR的Array-CGH 或其他微陣列芯片研究的文獻(xiàn)[2，7～31](包括MR人群研究和MR相關(guān)微陣列芯片研究回顧或綜述)。通過(guò)比對(duì)共發(fā)現(xiàn)461個(gè)CNVs， 涉及312個(gè)不同染色體條帶位點(diǎn)(圖5)，其中高發(fā)位點(diǎn)分別為22q11.2(19次)，Xp22.31(16次)，17p11.2(13次)，1p36.33(10次)，7q11.23(10次)，17q21.31(10次)，22q11.21(10次)，Xq28(10次)。本研究發(fā)現(xiàn)的CNVs中, 11/36個(gè)(30.6%)曾被既往MR微陣列研究文獻(xiàn)所報(bào)道。

圖5 MR相關(guān)CNVs在23條染色體上的分布

Fig 5 The distribution of CNVs related to MR on 23 chromosomes

Notes Rare CNVs from the literature review were mapped out according to position of 23 chromosomes (red/green). Red bar meant duplication and green meant deletion. Rare CNVs from our dataset are shown as black bars

2.4 1例非典型性PWS患兒的臨床表型 病例號(hào)211，男，2歲3個(gè)月，G3P2，足月順產(chǎn)，出生體重2 250 g，出生身長(zhǎng)不詳，生后喂養(yǎng)較困難，體重增長(zhǎng)緩慢，9個(gè)月會(huì)抬頭，1歲會(huì)坐，2歲會(huì)走，2歲會(huì)發(fā)“baba”和“mama”音，有驚厥史。父母否認(rèn)近親結(jié)婚、孕期藥物毒物接觸和家族性MR病史。查體：體重10.5 kg(<第1百分位，P1)，身長(zhǎng)80 cm(

圖6 非典型性PWS患兒面部表型和MRI所見(jiàn)

Fig 6 The facial characteristics and MRI in a patient with atypical PWS

Notes A,B:facial characteristics of patient 211 including flat face, sparse hair, hypertelorism and down-slanting eyes. Written consent to publish these images had been obtained from his legal guardian; C,D: MRI showed prominent bilateral frontal and temporal lobe sulcus, enlarged lateral ventricles

圖7 非典型性PWS患兒的Array-CGH結(jié)果和Refseq基因圖

Fig 7 The array-CGH data and scheme of Refseq gene in a patient with atypical PWS

Notes A: Array-CGH data showed the deletion in PWS/AS critical region of patient 211;B: Scheme of chromosome band and involved refseq genes in 15q11-13 (21219452-26208646). In the top panel, an ideogram showed deletion band of proximal chromosome 15q11.2-13. Bottom panel showed affected genes in this region. In middle panel, smaller black bar was represented for deleted region in patient 211, bigger black bar was represented for patient 232. The common candidate genes of PWS/AS includeSNRPN，NECDIN，SnRNAs，UBE3A

3 討論

3.1 Array-CGH在篩查MR/DD相關(guān)CNVs的診斷作用 Array-CGH屬于DNA微陣列技術(shù)一種，是在原有高分辨染色體CGH基礎(chǔ)上發(fā)展起來(lái)的分子遺傳學(xué)技術(shù)，其基本原理是用不同的熒光染料分別標(biāo)記待測(cè)和參考DNA樣本，等量混合后與微陣列玻片上的oligonucleotide探針進(jìn)行競(jìng)爭(zhēng)性雜交。該技術(shù)通過(guò)一次雜交實(shí)驗(yàn)就可以獲知整個(gè)基因組的CNVs，在染色體微結(jié)構(gòu)改變、標(biāo)記染色體來(lái)源的判定等方面具有明顯的診斷優(yōu)勢(shì)，可檢測(cè)出除基因突變或染色體平衡易位以外的幾乎所有基因組失衡。

目前，歐美發(fā)達(dá)國(guó)家已將DNA微陣列技術(shù)應(yīng)用于不明原因MR/DD的常規(guī)分子遺傳檢測(cè)，并進(jìn)入到患兒醫(yī)療保險(xiǎn)內(nèi)。國(guó)外眾多研究已證實(shí)在不明原因MR/DD患者中，10%～20%存在CNVs，并認(rèn)為這種基因組失衡是MR/DD，甚至包括神經(jīng)、精神疾患的致病原因之一[4，32，33]。另有遺傳學(xué)專家提出可將微陣列芯片取代常規(guī)染色體檢查，作為MR/DD患兒的首要檢查項(xiàng)目，以減少患兒就診費(fèi)用和時(shí)間[31]。隨著微陣列技術(shù)的不斷發(fā)展，從BAC array，cDNA array到最新發(fā)展的寡核苷酸芯片(oligo array)，從44 K到100 K再到244 K和1 M，微陣列芯片分辨率快速提高，即使50～100 K的微缺失或重復(fù)都能成功捕獲，這使得MR/DD患兒基因組失衡的陽(yáng)性檢出率極大提高。而早期BAC 芯片，通常100 kb片段僅覆蓋1個(gè)克隆，陽(yáng)性檢出率<10%[3]。本研究使用Agilent公司的Oligo 224 K芯片，陽(yáng)性檢出率為17.1%(19/111例),與國(guó)外研究報(bào)道大致相符[34]，這些結(jié)果極大豐富了MR/DD的臨床病因?qū)W研究，也說(shuō)明該芯片可用于不明原因MR/DD的病因診斷。

除Array-CGH外，定量PCR、MLPA技術(shù)以及SNP array 也可對(duì)基因組進(jìn)行拷貝數(shù)分析，定量PCR和MLPA技術(shù)由于涉及基因組特定位點(diǎn)，CNVs檢出能力無(wú)法與微陣列芯片相比。國(guó)外研究顯示，MLPA能發(fā)現(xiàn)5%～10%不明原因MR患兒存在亞端粒區(qū)域CNVs[35]。中國(guó)北京大學(xué)第一醫(yī)院兒科曾利用MLPA分析39例MR患兒的23條染色體亞端粒結(jié)構(gòu)，發(fā)現(xiàn)4例(10%)存在亞端?？截悢?shù)缺失或重復(fù)[5]。SNP array是另一種新發(fā)展的DNA微陣列，Affymetrix公司的SNP 6.0雜交芯片將SNP位點(diǎn)探針和已知CNVs探針結(jié)合，提高了分辨率和基因組CNVs的檢出能力。Bernardini等[36]利用SNP 6.0芯片平臺(tái)，重新對(duì)51個(gè)既往低分辨率芯片結(jié)果陰性的患者進(jìn)行研究，顯示6%患者存在3個(gè)可能致病的CNVs,且CNVs片段均>75 kb。此類芯片不僅可發(fā)現(xiàn)已知可能致病的CNVs，還可以分析雜合子丟失(loss of heterozygosity，LOH)和SNP位點(diǎn)信息。

3.2 MR/DD相關(guān)罕見(jiàn)CNVs的評(píng)估方法 隨著比較基因組學(xué)研究的快速發(fā)展，如何發(fā)現(xiàn)和評(píng)估罕見(jiàn)致病性CNVs已成為一個(gè)面臨挑戰(zhàn)的重要任務(wù)。目前，國(guó)外很多分子診斷實(shí)驗(yàn)室都針對(duì)各自的實(shí)驗(yàn)平臺(tái)，制定相應(yīng)的評(píng)估方法[30，31，34，37]，主要因素包括：①是否為新生CNVs；②是否在正常人群中有報(bào)道；③CNVs長(zhǎng)度是否足夠大；④是否在DECIPHER 或自己實(shí)驗(yàn)室的數(shù)據(jù)庫(kù)中有報(bào)道，且攜帶者具有MR/DD表型[4]。但由于患兒雙親取樣困難，因此臨床信息、DGV和DECIPHER數(shù)據(jù)庫(kù)比對(duì)顯得尤為重要。本研究中將“是否曾在不明原因MR/DD人群中有報(bào)道”加入評(píng)估標(biāo)準(zhǔn)中，因?yàn)橐恍┘韧芯坎](méi)有將自己發(fā)現(xiàn)的罕見(jiàn)CNVs數(shù)據(jù)載入DECIPHER數(shù)據(jù)庫(kù)。本研究結(jié)果顯示，6個(gè)CNVs未被DECIPHER數(shù)據(jù)庫(kù)報(bào)道。因此通過(guò)結(jié)合既往研究和美國(guó)波士頓兒童醫(yī)院Array-CGH數(shù)據(jù)庫(kù)，本研究最終將其中3個(gè)歸為可能的致病性CNVs(表1)，提高了MR/DD相關(guān)CNVs的診斷檢出率，也提示既往文獻(xiàn)回顧可作為MR/DD相關(guān)CNVs的一種評(píng)估方法。

3.3 非典型性PWS PWS/AS 是由于15q11-13區(qū)域上部分或全部印記基因簇發(fā)生突變、缺失、甲基化修飾異常或者單親二體所引起。如果母本基因在該區(qū)域甲基化導(dǎo)致基因表達(dá)沉默而父本基因出現(xiàn)突變、缺失、甲基化異?；騿斡H二體則導(dǎo)致PWS，如果父本基因在該區(qū)域甲基化導(dǎo)致基因表達(dá)沉默而母本基因出現(xiàn)突變、缺失、甲基化異?；騿斡H二體則導(dǎo)致AS。15q11-13區(qū)域父本基因缺失是PWS最常見(jiàn)的病因[38]；而母本基因異常則導(dǎo)致AS。本研究發(fā)現(xiàn)2例患兒(病例號(hào)232和211)存在PWS/AS關(guān)鍵區(qū)域缺失，其中1例(病例號(hào)232)缺失4 989 kb，涉及SNRPN、NECDIN、SnRNAs和UBE3A等重要印記基因簇，1例(病例號(hào)211)雖然缺失區(qū)域僅為2 098 kb，仍包含以上重要致病基因，從基因組特點(diǎn)可稱為非典型性PWS。

4 總結(jié)

本研究利用國(guó)際前沿的Array-CGH對(duì)中國(guó)人群中不明原因MR/DD患兒開(kāi)展與MR/DD相關(guān)的CNVs研究，結(jié)果顯示17.1%患兒攜帶可能與MR/DD相關(guān)的致病CNVs，其中已確定的部分罕見(jiàn)CNVs是中國(guó)人群中部分不明原因MR/DD患兒的發(fā)病原因之一。本研究同時(shí)發(fā)現(xiàn)，Array-CGH可幫助診斷某些與MR/DD相關(guān)但臨床不易察覺(jué)的非典型性綜合征，為部分不明原因的MR/DD患兒提供準(zhǔn)確的遺傳病因診斷。本研究作為轉(zhuǎn)化醫(yī)學(xué)研究的一項(xiàng)實(shí)踐，不僅可將這個(gè)新的技術(shù)平臺(tái)在中國(guó)及時(shí)推廣應(yīng)用于臨床實(shí)踐，加強(qiáng)和提高對(duì)不明原因MR/DD的分子診斷水平，而且對(duì)這些已發(fā)現(xiàn)的罕見(jiàn)CNVs區(qū)域所包括的許多基因可做進(jìn)一步的挖掘，從中發(fā)現(xiàn)關(guān)鍵的致病主基因，并深入研究其致病機(jī)制，實(shí)現(xiàn)轉(zhuǎn)化醫(yī)學(xué)B2B(from bench to bed)的目標(biāo)。

[1]Shevell M, Ashwal S, Donley D, et al. Practice parameter: evaluation of the child with global developmental delay: report of the Quality Standards Subcommittee of the American Academy of Neurology and The Practice Committee of the Child Neurology Society.Neurology,2003,60(3):367-380

[2]Shaffer LG,American College of Medical Genetics Professional Practice and Guidelines Committee. American College of Medical Genetics guideline on the cytogenetic evaluation of the individual with developmental delay or mental retardation.Genet Med,2005,7(9):650-654

[3]Shaw-Smith C, Redon R, Rickman L, et al. Microarray based comparative genomic hybridization (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet,2004,41(4):241-248

[4]Vissers LE, de Vries BB, Veltman JA.Genomic microarrays in mental retardation: from CNV to gene, from research to diagnosis.J Med Genet, 2010 in press

[5]Wu Y(吳曄),Jiang YW,Wang XZ，et al.Detection of subte-lomeric rearrangements in patients with idiopathic mental retardation/developmental delay.Chin J Pediatr(中華兒科雜志),2007,45(12):906-911

[6]Shen Y, Irons M, Miller DT, et al. Development of a focused oligonucleotide-array comparative genomic hybridization chip for clinical diagnosis of genomic imbalance. Clin Chem, 2007,53(12):2051-2059

[7]Vissers LE, de Vries BB, Osoegawa K, et al. Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. Am J Hum Genet,2003,73(6):1261-1270

[8]de Vries BB, Pfundt R, Leisink M, et al. Diagnostic genome profiling in mental retardation. Am J Hum Genet,2005,77(4):606-616

[9]Schoumans J, Ruivenkamp C, Holmberg E, et al. Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH). J Med Genet,2005，42(9):699-705

[10]Menten B, Maas N, Thienpont B, et al. Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports. J Med Genet,2006，43(8):625-633

[11]Rosenberg C, Knijnenburg J, Bakker E, et al. Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents. J Med Genet,2006，43(2):180-186

[12]Sharp AJ, Hansen S, Selzer RR, et al. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet,2006，3(9):1038-1042

[13]Aradhya S, Manning MA, Splendore A, et al. Whole-genome array-CGH identifies novel contiguous gene deletions and duplications associated with developmental delay, mental retardation, and dysmorphic features. Am J Med Genet A,2007，143A(13):1431-1441

[14]Baris HN, Tan WH, Kimonis VE, et al. Diagnostic utility of array-based comparative genomic hybridization in a clinical setting. Am J Med Genet A,2007,143(21):2523-2533

[15]Engels H, Brockschmidt A, Hoischen A, et al. DNA micro-array analysis identifies candidate regions and genes in unexplained mental retardation. Neurology,2007，68(10):743-750

[16]Fan YS, Jayakar P, Zhu H, et al. Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridi-zation. Hum Mutat,2007,28(11):1124-1132

[17]Lu X, Shaw CA, Patel A, et al.Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases.PLoS One, 2007,2(3):e327

[18]Shaffer LG, Bejjani BA, Torchia B, et al. The identification of microdeletion syndromes and other chromosome abnormalities: cytogenetic methods of the past, new technologies for the future. Am J Med Genet C Semin Med Genet, 2007，145C(4):335-345

[19]Thuresson AC, Bondeson ML, Edeby C, et al. Whole-genome array-CGH for detection of submicroscopic chromosomal imbalances in children with mental retardation. Cytogenet Genome Res, 2007，118(1):1-7

[20]Wagenstaller J, Spranger S, Lorenz-Depiereux B,et al. Copy-number variations measured by single-nucleotide-polymorphism oligonucleotide arrays in patients with mental retardation. Am J Hum Genet, 2007，81(4):768-779

[21]Pickering DL, Eudy JD, Olney AH, et al. Array-based comparative genomic hybridization analysis of 1176 consecutive clinical genetics investigations. Genet Med,2008,10(4):262-266

[22]Lybaek H, Meza-Zepeda LA, Kresse SH, et al. Array-CGH fine mapping of minor and cryptic HR-CGH detected genomic imbalances in 80 out of 590 patients with abnormal development. Eur J Hum Genet, 2008，16(11):1318-1328

[23]Nowakowska B, Stankiewicz P, Obersztyn E, et al. Application of metaphase HR-CGH and targeted Chromosomal Microarray Analyses to genomic characterization of 116 patients with mental retardation and dysmorphic features. Am J Med Genet A,2008，146A(18):2361-2369

[24]Krepischi-Santos AC, Vianna-Morgante AM, Jehee FS, et al. Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations. Cytogenet Genome Res,2006,115(3-4):254-261

[25]Friedman JM, Baross A, Delaney AD, et al. Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation. Am J Hum Genet, 2006, 79(3):500-513

[26]Koolen DA, Vissers LE, Pfundt R, et al. A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism. Nat Genet,2006,38(9):999-1001

[27]Lugtenberg D, de Brouwer AP, Kleefstra T, et al. Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH. J Med Genet,2006，43(4):362-370

[28]Shevell MI, Bejjani BA, Srour M, et al. Array comparative genomic hybridization in global developmental delay. Am J Med Genet B Neuropsychiatr Genet,2008,147B(7):1101-1108

[29]Sagoo GS, Butterworth AS, Sanderson S, et al. Array CGH in patients with learning disability (mental retardation) and congenital anomalies: updated systematic review and meta-analysis of 19 studies and 13,926 subjects. Genet Med,2009,11(3):139-146

[30]McMullan DJ, Bonin M, Hehir-Kwa JY, et al. Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: a multicenter study. Hum Mutat,2009，30(7):1082-1092

[31]Gijsbers AC, Lew JY, Bosch CA,et al. A new diagnostic workflow for patients with mental retardation and/or multiple congenital abnormalities: test arrays first. Eur J Hum Genet, 2009，17(11):1394-1402

[32]St Clair D.Copy number variation and schizophrenia.Schizophr Bull,2009,35(1):9-12

[33]Weiss LA, Shen Y, Korn JM, et al. Association between microdeletion and microduplication at 16p11.2 and autism. N Engl J Med,2008, 358(7):667-675

[34]Hochstenbach R, van Binsbergen E, Engelen J,et al.Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands.Eur J Med Genet,2009,52(4):161-169

[35]Koolen DA,Nillesen WM,Versteeg MH,et al.Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA).J Med Genet,2004,41(12):892-899

[36]Bernardini L, Alesi V, Loddo S,et al.High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?Eur J Hum Genet, 2010,18(2):178-185

[37]Koolen DA, Pfundt R, de Leeuw N, et al. Genomic microarrays in mental retardation: a practical workflow for diagnostic applications. Hum Mutat,2009，30(3):283-292

[38]Prader-Willi syndrome association.http://www.pwsausa.org/syndrome/index.htm