15q11.2-13.2微重复四倍体综合征1例并文献复习

李小燕 陈 倩 谢 华 王立文 陈晓丽 李尔珍 钟建民



·论著·

15q11.2-13.2微重复四倍体综合征1例并文献复习

李小燕1陈 倩2谢 华3王立文2陈晓丽3李尔珍2钟建民1

15q11.2-13.2微重复四倍体综合征； 发育迟缓； 孤独症； 额外标记染色体； 微阵列比较基因组杂交； 荧光原位杂交技术

1 病例资料

女， 2岁，因“自幼发育落后”于2014年8月13日就诊于首都儿科研究所。患儿系G2P2，因母亲妊娠期高血压于孕38周剖宫娩出，围生期无窒息缺氧史，出生体重2 700 g，身长49 cm。肌张力低下，5月龄抬头。尚不能独站、行走及有意识言语。能逗笑，不认人，不能交流，缺乏目光对视。未见抽搐，无侵略性行为。能跟着拍手和摇头，对音乐、汽车声音及发光的物体感兴趣，喜欢看手玩手，常有双手扭转，喜欢独处，不能使用任何工具。

查体：身长90 cm(P75)，体重14 kg(P90)。双眼无神，鼻梁低平，外侧眼角下垂、内眦赘皮(图1)。

图1 本文患儿正侧面照

Fig 1 Clinical appearance of the patient

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

母孕期未补充叶酸及钙。母亲本次妊娠年龄32岁，父亲37岁。患儿姐姐未见异常表型。

辅助检查： 脑电图、头颅MRI、血生化、甲状腺功能检查、血尿遗传代谢病筛查均未见异常。2次外院常规染色体核型检查(320带)分别为47,XX,+22和47,XX,+21。中国孤独症评量表(CARS)得分48分(诊断界值为30分)。0～6岁儿童神经心理发育测试量表(DST)发育商得分36分。

患儿监护人签署知情同意书后，采集患儿及其父母外周静脉血(肝素抗凝)行高分辨率染色体核型分析(400-550带)。取0.2 mL接种于2.5mL培养基中，37℃培养72 h，加秋水仙素继续培养3 h，离心抽取上清液收获细胞，滴片，在显微镜下观察细胞显带，合格后放入干燥箱干燥。干燥后进行染色体处理、风干和盖玻片封片。选择40个以上分裂相作众数分析。再用油镜选择染色体较长、带纹较清晰及交叉点低的分裂相行核型分析，每个样本分析至少20个核型。异常克隆标准为2个细胞具有相同的额外染色体或结构重排，或至少3个细胞至少丢失1个相同的染色体。核型异常按《人类细胞遗传学国际命名体制(ISCN 2013)》标准判定[1]。高分辨染色体核型分析显示，患儿较正常染色体核型多1条额外标记染色体(SMC)，SMC可能来源于随体型D群染色体(13号，14号，15号染色体)或G群染色体(21号，22号染色体)，核型为47，XX，+mar dn(图2)。患儿父亲高分辨率染色体核型分析提示为46,XY，母亲为46,XX。提示患儿携带的SMC为新生突变。

图2 本文患儿高分辨染色体核型分析结果(400-550带)

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

Notes The figure showed SMC marked by the red arrow

采集患儿外周静脉血2 mL(EDTA抗凝)，并用酚-氯仿法抽提基因组DNA。获得吸光度值和浓度后，取1 μg DNA，37℃消化2.5 h(Alu Ⅰ和Rsa Ⅰ联合消化)；用SureTag DNA Labeling Kit(Agilent公司)行荧光标记(Cy5标记对照标本，Cy3标记先证者标本)，37℃ 2 h后终止反应；用MieroCon YM-30(Agilent公司)纯化荧光标记DNA后，将对照DNA和患儿DNA等量混合后加入杂交体系，95℃变性，37℃ 0.5 h，上样到Agilent 4×180K CNV+SNP定制芯片(Agilent公司)，65℃杂交炉孵育36 h后先用洗脱液1洗脱5 min，再用洗脱液2洗脱1 min，行微阵列比较基因组杂交(array-CGH)扫描，采用Feature Extraction 9.0进行数据提取，DNA analystic 5.0软件进行CNVs分析。图3显示，本文患儿15q11.2-13.2区域存在8.0 Mb的微重复(hg19)，且log2>1.25，提示该区域拷贝数可能为4倍体。该区域为Prader-Willi/Angelman综合征的典型致病区域，属临床致病性微缺失。

结合array-CGH结果，采用商业化Prader-Willi/Angelman综合征关键区域探针(UBE3A，D15S10：15q11.2红色)和15号染色体识别用控制探针(D15Z1:15p11.2蓝色，PML:15q22 绿色)直接杂交。实验步骤按照说明书，包括：培养、秋水仙素处理、低渗处理37℃水浴、卡诺氏固定、细胞滴下、载玻片预处理、染色体DNA变性、杂化(使用VYSIS探针)、对比染色和获取图像。

FISH结果可见SMC包括着丝粒信号D15Z1(蓝)2个，基因UBE3A/D15S10(红)信号2个(图4)。结合array-CGH结果，提示患儿SMC为15号染色体以长臂q13为断裂点的双着丝粒异常染色体，即为SMC(15)。FISH结果证实array-CGH 筛查出基因组微重复log数值>1.25的区域存在4个基因组拷贝。

图3 本文患儿array-CGH结果

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

图4 本文患儿FISH结果

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

根据ISCN 2013[1]，患儿的染色体核型定义为：47, XX, +mar dn.ish dic(15;15)(q13;q13) (D15Z1++,UBE3A/D15S10++).arr15q11.2-q13.2(22684529-30730543)×4.de novo，即为15q11.2-13.2微重复四倍体。

2 文献复习

3 讨论

本文患儿无法交流，缺乏目光对视。能跟着拍手和摇头，对音乐、汽车声音及发光的物体感兴趣，喜欢看手玩手，常有双手扭转，喜欢独处，不能使用任何工具。CARS评分48分、DST评分36分，符合DSM-Ⅳ中发育迟缓和孤独症的诊断标准。

本文患儿高分辨染色体检查存在核型异常，按ISCN 2013标准异常染色体为SMC。SMC指细胞核内46条正常染色体核型以外的染色体，是引起基因组拷贝数增加的重要原因之一。SMC多为染色体易位所致，约64%的复杂SMC来源于父母染色体平衡易位，36%为新生突变[14,15]。在活产婴儿中SMC发生率为0.014%～0.072%，智力缺陷患者中SMC的发生率为0.433%[16,17]。SMC携带者表型可从正常至严重异常，影响表型的因素有：①常染色体DNA所包含的内容；②嵌合程度；③SMC的单亲二倍体一致性[18,19]。本文患儿父母高分辨染色体核型分析正常，提示患儿SMC属于新生突变，文献报道SMC如为新生突变，则表型异常者占14%～30%[15]。

表1 既往报道和本研究中15q11.2-13.2微重复综合征临床表型[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大小一般与G组20、21、22号染色体相似或更小，最常见来源于近端着丝粒染色体，大部分源于15号染色体(约60%)或22号染色体(约9%)，多为母源性[20～22]。常规染色体核型检测一般无法区别21/22三体和SMC，容易误诊，本文患儿在外院曾误诊为21/22三体综合征，由于临床表型与基因型不符，遂再次进行高分辨染色体核型分析才发现为SMC，因此SMC需依靠高分辨染色体核型分析和(或)分子遗传学方法(如FISH、array-CGH)才能确诊[23,24]。21三体综合征表型主要为智力落后、特殊面容、生长发育迟缓，并可伴有多种畸形；特殊面容包括表情呆滞、眼裂小、眼距宽、双眼外眦上斜，可有内眦赘皮；鼻梁低平、外耳小、硬腭窄小；常张口伸舌，流涎多。22三体综合征主要临床表型为智力低下，伴多系统畸形，如单侧或双侧眼缺损、外侧眼角下垂、耳前畸形、心脏及肾脏畸形、肝门瘘管或(和)闭锁[25]。本文患儿有生长发育迟缓、孤独症样表现、表情呆滞、内眦赘皮，但无眼裂小、眼距宽、外眦上斜、张口伸舌、流涎和多种畸形，表型不符合21/22三体综合征。因此，当临床发现染色体核型分析结果与临床表型不相符时，需要进一步行高分辨染色体核型分析、array-CGH和FISH检测，明确核型异常的类型，避免诊误。

Array-CGH分析发现本文患儿的SMC为15q11.2-13.2区域存在8.0 Mb的微重复(log>1.25)，提示该区域拷贝数可能为四倍体。拷贝数变异(CNV)由基因组重排所致，主要包括基因组片段的微缺失和微重复，片段大小为1 kb至Mb级。现有研究发现15.0%～21.4%的儿童精神心理发育异常由CNV所致[26,27]。FISH检测进一步明确SMC来源于15号染色体，且为四倍体。15号染色体长臂由于存在低拷贝重复序列，结构不稳定，倾向于各种基因重组，包括微缺失、微重复和易位。SMC(15)导致的15q微重复的重复倍数有3、4、5和6倍[5,12]。SMC(15)根据是否包含Prader-Willi/Angelman综合征的关键区域分为2组：一组为小片段的、除男性不孕外不导致其他临床表型；一组为大片段的、导致相关临床表现[10]。本文病例为15q11.2-13.2四倍体及双着丝粒组成的大片段的、导致相关临床表现的SMC(15)。根据基因组CNV起止点位置，15号染色体长臂可以分为5个断点(BP1-BP5)。根据断点，可将15号染色体长臂CNV分为3类：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]根据断点将微重复分为Ⅰ类和Ⅱ类，Ⅰ类为BP1-BP3，Ⅱ类为BP2-BP3，本文患儿为BP1-BP3，属于Ⅰ类。文献中迄今仅有16例报道有明确的起止点，根据断点分类均为Ⅰ类微重复[5,11,12]。

总之，对于SMC携带者，单纯依靠高分辨染色体核型检测，无法确定其基因组来源及拷贝数量化。如常规染色体核型结果和临床表型不符合，应行array-CGH检测确诊基因拷贝数变异位点；对于array-CGH所发现的基因组微重复，Log数值高于1.25～1.30，应行FISH、real-time PCR等以明确基因组拷贝数目、重组结构特点。这对遗传咨询和临床干预措施的制定均有重要的意义。

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(本文编辑：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 江西省儿童医院神经内科 南昌，330006；2 首都儿科研究所附属儿童医院神经内科 北京，100020；3 首都儿科研究所北京市儿童发展和营养组学重点实验室 北京，100020

钟建民，E-mail:zhongjm@163.com; 李尔珍，E-mail:13693355316@163.com

10.3969/j.issn.1673-5501.2015.04.011

2015-07-05

2015-07-17)