李小燕 陳 倩 謝 華 王立文 陳曉麗 李爾珍 鐘建民

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·論著·

15q11.2-13.2微重復(fù)四倍體綜合征1例并文獻(xiàn)復(fù)習(xí)

李小燕1陳 倩2謝 華3王立文2陳曉麗3李爾珍2鐘建民1

15q11.2-13.2微重復(fù)四倍體綜合征； 發(fā)育遲緩； 孤獨(dú)癥； 額外標(biāo)記染色體； 微陣列比較基因組雜交； 熒光原位雜交技術(shù)

1 病例資料

女， 2歲，因“自幼發(fā)育落后”于2014年8月13日就診于首都兒科研究所?；純合礕2P2，因母親妊娠期高血壓于孕38周剖宮娩出，圍生期無窒息缺氧史，出生體重2 700 g，身長49 cm。肌張力低下，5月齡抬頭。尚不能獨(dú)站、行走及有意識(shí)言語。能逗笑，不認(rèn)人，不能交流，缺乏目光對(duì)視。未見抽搐，無侵略性行為。能跟著拍手和搖頭，對(duì)音樂、汽車聲音及發(fā)光的物體感興趣，喜歡看手玩手，常有雙手扭轉(zhuǎn)，喜歡獨(dú)處，不能使用任何工具。

查體：身長90 cm(P75)，體重14 kg(P90)。雙眼無神，鼻梁低平，外側(cè)眼角下垂、內(nèi)眥贅皮(圖1)。

圖1 本文患兒正側(cè)面照

Fig 1 Clinical appearance of the patient

Notes She presented negative face,downslanting of the palpebral fissures, flat nasal bridge and epicanthic folds

母孕期未補(bǔ)充葉酸及鈣。母親本次妊娠年齡32歲，父親37歲?；純航憬阄匆姰惓１硇?。

輔助檢查： 腦電圖、頭顱MRI、血生化、甲狀腺功能檢查、血尿遺傳代謝病篩查均未見異常。2次外院常規(guī)染色體核型檢查(320帶)分別為47,XX,+22和47,XX,+21。中國孤獨(dú)癥評(píng)量表(CARS)得分48分(診斷界值為30分)。0～6歲兒童神經(jīng)心理發(fā)育測(cè)試量表(DST)發(fā)育商得分36分。

患兒監(jiān)護(hù)人簽署知情同意書后，采集患兒及其父母外周靜脈血(肝素抗凝)行高分辨率染色體核型分析(400-550帶)。取0.2 mL接種于2.5mL培養(yǎng)基中，37℃培養(yǎng)72 h，加秋水仙素繼續(xù)培養(yǎng)3 h，離心抽取上清液收獲細(xì)胞，滴片，在顯微鏡下觀察細(xì)胞顯帶，合格后放入干燥箱干燥。干燥后進(jìn)行染色體處理、風(fēng)干和蓋玻片封片。選擇40個(gè)以上分裂相作眾數(shù)分析。再用油鏡選擇染色體較長、帶紋較清晰及交叉點(diǎn)低的分裂相行核型分析，每個(gè)樣本分析至少20個(gè)核型。異常克隆標(biāo)準(zhǔn)為2個(gè)細(xì)胞具有相同的額外染色體或結(jié)構(gòu)重排，或至少3個(gè)細(xì)胞至少丟失1個(gè)相同的染色體。核型異常按《人類細(xì)胞遺傳學(xué)國際命名體制(ISCN 2013)》標(biāo)準(zhǔn)判定[1]。高分辨染色體核型分析顯示，患兒較正常染色體核型多1條額外標(biāo)記染色體(SMC)，SMC可能來源于隨體型D群染色體(13號(hào)，14號(hào)，15號(hào)染色體)或G群染色體(21號(hào)，22號(hào)染色體)，核型為47，XX，+mar dn(圖2)?；純焊赣H高分辨率染色體核型分析提示為46,XY，母親為46,XX。提示患兒攜帶的SMC為新生突變。

圖2 本文患兒高分辨染色體核型分析結(jié)果(400-550帶)

Fig 2 The result of high-resolution karyotype analysis (400-550 bands)

Notes The figure showed SMC marked by the red arrow

采集患兒外周靜脈血2 mL(EDTA抗凝)，并用酚-氯仿法抽提基因組DNA。獲得吸光度值和濃度后，取1 μg DNA，37℃消化2.5 h(Alu Ⅰ和Rsa Ⅰ聯(lián)合消化)；用SureTag DNA Labeling Kit(Agilent公司)行熒光標(biāo)記(Cy5標(biāo)記對(duì)照標(biāo)本，Cy3標(biāo)記先證者標(biāo)本)，37℃ 2 h后終止反應(yīng)；用MieroCon YM-30(Agilent公司)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，95℃變性，37℃ 0.5 h，上樣到Agilent 4×180K CNV+SNP定制芯片(Agilent公司)，65℃雜交爐孵育36 h后先用洗脫液1洗脫5 min，再用洗脫液2洗脫1 min，行微陣列比較基因組雜交(array-CGH)掃描，采用Feature Extraction 9.0進(jìn)行數(shù)據(jù)提取，DNA analystic 5.0軟件進(jìn)行CNVs分析。圖3顯示，本文患兒15q11.2-13.2區(qū)域存在8.0 Mb的微重復(fù)(hg19)，且log2>1.25，提示該區(qū)域拷貝數(shù)可能為4倍體。該區(qū)域?yàn)镻rader-Willi/Angelman綜合征的典型致病區(qū)域，屬臨床致病性微缺失。

結(jié)合array-CGH結(jié)果，采用商業(yè)化Prader-Willi/Angelman綜合征關(guān)鍵區(qū)域探針(UBE3A，D15S10：15q11.2紅色)和15號(hào)染色體識(shí)別用控制探針(D15Z1:15p11.2藍(lán)色，PML:15q22 綠色)直接雜交。實(shí)驗(yàn)步驟按照說明書，包括：培養(yǎng)、秋水仙素處理、低滲處理37℃水浴、卡諾氏固定、細(xì)胞滴下、載玻片預(yù)處理、染色體DNA變性、雜化(使用VYSIS探針)、對(duì)比染色和獲取圖像。

FISH結(jié)果可見SMC包括著絲粒信號(hào)D15Z1(藍(lán))2個(gè)，基因UBE3A/D15S10(紅)信號(hào)2個(gè)(圖4)。結(jié)合array-CGH結(jié)果，提示患兒SMC為15號(hào)染色體以長臂q13為斷裂點(diǎn)的雙著絲粒異常染色體，即為SMC(15)。FISH結(jié)果證實(shí)array-CGH 篩查出基因組微重復(fù)log數(shù)值>1.25的區(qū)域存在4個(gè)基因組拷貝。

圖3 本文患兒array-CGH結(jié)果

Fig 3 The result of the array-CGH

Notes A: 8.0 Mb duplication in the 15q11.2-13.2 region (chr15:22684529-30730543, 8.0 Mb, hg19) was found, log2 ratio >1.25. The region contained 217 genes, includingUBE3A,GABRB3,GABRA5,GABRG3,ABPA2,CHRFAM7AandCHRNA7 associated with the disease

圖4 本文患兒FISH結(jié)果

Fig 4 The result of FISH

Notes The red arrow showed SMC(15), containing 2 blue signals, 2 red signals and the white arrows showed two normal chromosome 15 including a blue signal,a red signal and a green signal

根據(jù)ISCN 2013[1]，患兒的染色體核型定義為：47, XX, +mar dn.ish dic(15;15)(q13;q13) (D15Z1++,UBE3A/D15S10++).arr15q11.2-q13.2(22684529-30730543)×4.de novo，即為15q11.2-13.2微重復(fù)四倍體。

2 文獻(xiàn)復(fù)習(xí)

3 討論

本文患兒無法交流，缺乏目光對(duì)視。能跟著拍手和搖頭，對(duì)音樂、汽車聲音及發(fā)光的物體感興趣，喜歡看手玩手，常有雙手扭轉(zhuǎn)，喜歡獨(dú)處，不能使用任何工具。CARS評(píng)分48分、DST評(píng)分36分，符合DSM-Ⅳ中發(fā)育遲緩和孤獨(dú)癥的診斷標(biāo)準(zhǔn)。

本文患兒高分辨染色體檢查存在核型異常，按ISCN 2013標(biāo)準(zhǔn)異常染色體為SMC。SMC指細(xì)胞核內(nèi)46條正常染色體核型以外的染色體，是引起基因組拷貝數(shù)增加的重要原因之一。SMC多為染色體易位所致，約64%的復(fù)雜SMC來源于父母染色體平衡易位，36%為新生突變[14,15]。在活產(chǎn)嬰兒中SMC發(fā)生率為0.014%～0.072%，智力缺陷患者中SMC的發(fā)生率為0.433%[16,17]。SMC攜帶者表型可從正常至嚴(yán)重異常，影響表型的因素有：①常染色體DNA所包含的內(nèi)容；②嵌合程度；③SMC的單親二倍體一致性[18,19]。本文患兒父母高分辨染色體核型分析正常，提示患兒SMC屬于新生突變，文獻(xiàn)報(bào)道SMC如為新生突變，則表型異常者占14%～30%[15]。

表1 既往報(bào)道和本研究中15q11.2-13.2微重復(fù)綜合征臨床表型[n/N(%)]
Tab 1 Clinical characteristics of 15q11.2-13.2 microduplication syndrome in the literatures and the case reported in this paper[n/N(%)]

SMC大小一般與G組20、21、22號(hào)染色體相似或更小，最常見來源于近端著絲粒染色體，大部分源于15號(hào)染色體(約60%)或22號(hào)染色體(約9%)，多為母源性[20～22]。常規(guī)染色體核型檢測(cè)一般無法區(qū)別21/22三體和SMC，容易誤診，本文患兒在外院曾誤診為21/22三體綜合征，由于臨床表型與基因型不符，遂再次進(jìn)行高分辨染色體核型分析才發(fā)現(xiàn)為SMC，因此SMC需依靠高分辨染色體核型分析和(或)分子遺傳學(xué)方法(如FISH、array-CGH)才能確診[23,24]。21三體綜合征表型主要為智力落后、特殊面容、生長發(fā)育遲緩，并可伴有多種畸形；特殊面容包括表情呆滯、眼裂小、眼距寬、雙眼外眥上斜，可有內(nèi)眥贅皮；鼻梁低平、外耳小、硬腭窄??；常張口伸舌，流涎多。22三體綜合征主要臨床表型為智力低下，伴多系統(tǒng)畸形，如單側(cè)或雙側(cè)眼缺損、外側(cè)眼角下垂、耳前畸形、心臟及腎臟畸形、肝門瘺管或(和)閉鎖[25]。本文患兒有生長發(fā)育遲緩、孤獨(dú)癥樣表現(xiàn)、表情呆滯、內(nèi)眥贅皮，但無眼裂小、眼距寬、外眥上斜、張口伸舌、流涎和多種畸形，表型不符合21/22三體綜合征。因此，當(dāng)臨床發(fā)現(xiàn)染色體核型分析結(jié)果與臨床表型不相符時(shí)，需要進(jìn)一步行高分辨染色體核型分析、array-CGH和FISH檢測(cè)，明確核型異常的類型，避免診誤。

Array-CGH分析發(fā)現(xiàn)本文患兒的SMC為15q11.2-13.2區(qū)域存在8.0 Mb的微重復(fù)(log>1.25)，提示該區(qū)域拷貝數(shù)可能為四倍體?？截悢?shù)變異(CNV)由基因組重排所致，主要包括基因組片段的微缺失和微重復(fù)，片段大小為1 kb至Mb級(jí)。現(xiàn)有研究發(fā)現(xiàn)15.0%～21.4%的兒童精神心理發(fā)育異常由CNV所致[26,27]。FISH檢測(cè)進(jìn)一步明確SMC來源于15號(hào)染色體，且為四倍體。15號(hào)染色體長臂由于存在低拷貝重復(fù)序列，結(jié)構(gòu)不穩(wěn)定，傾向于各種基因重組，包括微缺失、微重復(fù)和易位。SMC(15)導(dǎo)致的15q微重復(fù)的重復(fù)倍數(shù)有3、4、5和6倍[5,12]。SMC(15)根據(jù)是否包含Prader-Willi/Angelman綜合征的關(guān)鍵區(qū)域分為2組：一組為小片段的、除男性不孕外不導(dǎo)致其他臨床表型；一組為大片段的、導(dǎo)致相關(guān)臨床表現(xiàn)[10]。本文病例為15q11.2-13.2四倍體及雙著絲粒組成的大片段的、導(dǎo)致相關(guān)臨床表現(xiàn)的SMC(15)。根據(jù)基因組CNV起止點(diǎn)位置，15號(hào)染色體長臂可以分為5個(gè)斷點(diǎn)(BP1-BP5)。根據(jù)斷點(diǎn)，可將15號(hào)染色體長臂CNV分為3類：①15q11.2(BP1-BP2，chr15:22698322-23217655)；②15q11.2-q13.1(BP2-BP3，chr15:23707400-28520300)；③15q13.2-13.3(BP4-BP5，chr15: 30931844- 32510863)。Urraca 等[4]根據(jù)斷點(diǎn)將微重復(fù)分為Ⅰ類和Ⅱ類，Ⅰ類為BP1-BP3，Ⅱ類為BP2-BP3，本文患兒為BP1-BP3，屬于Ⅰ類。文獻(xiàn)中迄今僅有16例報(bào)道有明確的起止點(diǎn)，根據(jù)斷點(diǎn)分類均為Ⅰ類微重復(fù)[5,11,12]。

總之，對(duì)于SMC攜帶者，單純依靠高分辨染色體核型檢測(cè)，無法確定其基因組來源及拷貝數(shù)量化。如常規(guī)染色體核型結(jié)果和臨床表型不符合，應(yīng)行array-CGH檢測(cè)確診基因拷貝數(shù)變異位點(diǎn)；對(duì)于array-CGH所發(fā)現(xiàn)的基因組微重復(fù)，Log數(shù)值高于1.25～1.30，應(yīng)行FISH、real-time PCR等以明確基因組拷貝數(shù)目、重組結(jié)構(gòu)特點(diǎn)。這對(duì)遺傳咨詢和臨床干預(yù)措施的制定均有重要的意義。

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(本文編輯：丁俊杰)

A case of 15q11.2-13.2 tetraploid microduplication syndrome and literature review

LIXiao-yan1,CHENQian2,XIEHua3,WANGLi-wen2,CHENXiao-li3,LIEr-zhen2,ZHONGJian-min1

(1DepartmentofPediatricNeurology,JiangxiChildren′sHospital,Nanchang330006; 2DepartmentofNeurology,AffiliatedChildren′sHospitalofCapitalInstituteofPediatrics,Beijing100020; 3BeijingMunicipalKeyLaboratoryofChildDevelopmentandNutriomics,CapitalInstituteofPediatrics,Beijing100020,China)

ZHONG Jian-min，E-mail:zhongjm@163.com；LI Er-zhen，E-mail: 13693355316@163.com

ObjectiveTo identify the underlying genetic causes with multiple molecular genetic techniques in a female patient with neuro-developmental delay and autism, who has ever been diagnosed as 21/22 trisomy with conventional karyotype analysis.MethodsThe peripheral blood was collected from the patient and her parents. Genomic DNA was extracted by phenol-chloroform method. The high-resolution karyotype analysis (400-550 bands) was performed to check the chromosome′s number and structure, and the array comparative genomic hybridization (array-CGH) was used to detect the whole genomic copy number variation.The fluorescent in situ hybridization was employed to localize and quantify the abnormal genomic copy numbers.ResultsA 2-year-old girl suffered from neuro-developmental delay and autism with downslanting of the palpebral fissures and epicanthic folds. The result of conventional karyotype analysis (320 bands) was 47,XX,+22 or 47,XX,+21.The high-resolution karyotype analysis (400-550 bands) detected a supernumerary marker chromosome(SMC) and her karyotype was 47,XX,+mar dn, which had ever been misdiagnosed as 21/22 trisomy.Her parents′ karyotype was 46,XY and 46,XX, respectively. The SMC was de novo. About 8.0 Mb duplication in the 15q11.2-13.2 region (chr15:22684529-30730543, 8.0 Mb,hg19) was found in the patient by array-CGH. FISH confirmed that the SMC was originated from chromosome 15, and consisted of two copies of the centromerics and 15q11.2-13.2 interval. The 15q11.2-13.2 tetraploid microduplication syndrome or Idic(15) syndrome was established after all. A literature review of the clinical phenotypes of the 15q11.2-13.2 microduplication was performed, which showed that intellectual disability/ developmental retardation, hypotpnia, autism/autism-like symptoms and epilepsy were the key clinical phenotypes in the Idic(15) syndrome.ConclusionThe de novo tetrasomy 15q11.2-13.2 is a genetic basis for neuro-developmental delay and autism in this case. The array-CGH can detect genomic micro-imbalance quickly and precisely.

15q11.2-q13.2 tetraploid microduplication syndrome; Neuro-developmental delay; Autism; Supernumerary marker chromosomes; Array comparative genomic hybridization; Fluorescent in situ hybridization

1 江西省兒童醫(yī)院神經(jīng)內(nèi)科 南昌，330006；2 首都兒科研究所附屬兒童醫(yī)院神經(jīng)內(nèi)科 北京，100020；3 首都兒科研究所北京市兒童發(fā)展和營養(yǎng)組學(xué)重點(diǎn)實(shí)驗(yàn)室 北京，100020

鐘建民，E-mail:zhongjm@163.com; 李爾珍，E-mail:13693355316@163.com

10.3969/j.issn.1673-5501.2015.04.011

2015-07-05

2015-07-17)