综合应用分子细胞遗传学技术检测一例染色体微小易位

目的 综合应用分子细胞遗传学技术对1例染色体微小易位的病例进行检测.方法 按常规制备染色体,G显带进行核型分析,并先后进行光谱核型分析(spectral karyotyping,SKY),染色体涂染,双色荧光原位杂交技术(fluorescence in situ hybridisation,FISH)检测,亚端粒探针F...     

应用光谱核型分析技术诊断复杂染色体畸变

目的探讨光谱核型分析技术（spectral karyotyping，SKY）在诊断复杂染色体畸变中的应用价值。方法选取4例常规G显带染色体核型分析未能确诊的染色体畸变病例，按SKY操作常规进行制片杂交，并通过相应的计算机软件分析结果。结果通过SKY技术，明确1例涉及3条染色体复杂易位病例的诊断；协助2例不明来源衍生染色体诊断为染色体自身的部分重复；但对染色体自身倒位和染色体畸变断裂点的诊断帮助不大。结论SKY技术对于诊断复杂的染色体易位或重排、微小的染色体结构畸变以及不明来源的衍牛染色体等方面有较大的优越性．是常规染仁．体核型分析的有益补充。     

230例身材矮小患儿染色体核型分析

目的 通过对230例身材矮小患儿进行染色体核型分析,探讨身材矮小与染色体异常的关系.方法 对230例身材矮小患儿进行G显带染色体核型分析,并结合Q显带、C显带及荧光原位杂交等方法对异常染色体核型进行检测.结果 230例身材矮小患儿中染色体核型异常有23例,异常比例为10％.其中,Turner综合征核型18例,环状染色体3例(分别为4号、6号和15号环状染色体),其余两例分别为5号染色体倒位及10号染色体短臂三体.结论 染色体异常是引起身材矮小的重要原因之一,应用G带、Q带、C带及荧光原位杂交技术,精确诊断患者的染色体核型,可为临床的诊断和治疗提供医学遗传学依据.     

长沙地区24例染色体平衡易位的临床分析与遗传咨询

目的探讨染色体易位的遗传效应及染色体易位的临床分析和遗传咨询。方法采集外周血、羊水、脐血细胞培养,常规G显带技术进行核型分析。结果 2747例受检者中,共检出染色体平衡易位24例,其中相互易位10例,罗伯逊易位9例,还有羊水培养中5例相互易位的嵌合体。结论平衡易位携带是导致流产、死胎、死产、生育畸形儿的重要原因,要加强染色体平衡易位携带者的检出,做好携带者及家属的遗传咨询。     

应用单核苷酸多态芯片技术产前诊断四例胎儿新发染色体变异

目的 探讨单核苷酸多态性芯片(single nucleotide polymorphisms array,SNP-array)技术在新发染色体变异产前诊断中的应用价值.方法 选取产前诊断G显带染色体核型为新发平衡易位或微小额外标记染色体的4名孕妇,知情同意抽取胎儿脐带血DNA,按照标准的微阵列操作手册进行杂交、洗涤及全基因组扫描,扫描数据通过相应的计算机软件进行分析.结果 例1未发现致病性拷贝数改变;例2嵌合的微小额外标记染色体来源于4号染色体,包含约9Mb的重复;例3除了遗传了来源于母亲的两条平衡易位染色体,还出现了两条新发的易位染色体,但SNP-array未发现致病性拷贝数改变;例4的G显带显示的两条"平衡"易位染色体实为不平衡畸变:1号染色体25Mb的重复和9号染色体17Mb的缺失.例1和例3出生后随访智力体格发育均未见异常.结论 SNP-array能够在DNA水平上甄别胎儿新发"平衡"易位或微小额外标记染色体的致病性,在产前诊断中不失为传统染色体核型分析的重要补充.     

七例产前诊断的标记染色体及复杂染色体畸变的光谱核型分析

目的 探讨光谱核型分析( spectral karyotyping,SKY)结合荧光原位杂交(fluorescence in situ hybridization,FISH)及传统核型分析技术在产前诊断标记染色体及复杂染色体畸变中的应用.方法 对产前诊断中常规G显带分析发现的5例标记染色体以及2例复杂染色体畸变的胎儿样本进行SKY分析,必要时应用FISH技术进一步鉴定或采用C显带、N显带技术进行辅助诊断,并分析胎儿产前超声检查、生后随访或病理解剖结果.结果 5例标记染色体的病例中2例为大的标记染色体,3例为中等大小标记染色体;1例胎儿为父源性遗传,4例为新发突变.用SKY分析发现2例为非近端着丝粒来源(分别为4号、9号染色体),2例为近端着丝粒来源(分别来自22号、21号染色体),1例为X染色体来源.3例经FISH检查证实了SKY分析结果.5例标记染色体的胎儿4例终止妊娠,1例父源性遗传者足月分娩,生后随访1年未见异常.2例产前诊断为复杂染色体畸变的胎儿,其中1例经常规G显带分析为不明来源的衍生染色体,SKY诊断为8号染色体自身部分重复;另1例经SKY诊断为2号与6号染色体易位的胎儿足月分娩,随访6个月时有生长发育迟缓.结论 应用SKY结合FISH及传统的核型分析可对产前诊断中难以确定来源的标记染色体及复杂染色体畸变作出诊断,结合超声波检查结果,可更好地为临床咨询提供指导.     

一个涉及1号和7号染色体插入易位家系的鉴定

目的 确定一个有反复流产史且常规G显带发现有7q末端缺失病例的核型,探讨染色体末端区域插入易位的形成机理。方法 应用显微切割制备的7号特异性全染色体探针和7q亚端粒（7q36→qter)探针,与病例的中期分裂相进行荧光原位杂交（fluorescence in situ hybridization,FISH)。结果 发现了该病例为常规G显带难以妈现的1号和7号染色体之间的插入易位,7q36→qter区域没有插入到1号染色体中,其异常核型来源于其母亲。结论 为染色体末端区域的插入易位仍然为一个三断裂重排。细胞遗传学上见到的末端缺失为中间缺失提供了实验证据,FISH与显微切割技术相结合。是检出染色体微小结构异常的一个强有力的工具。     

多重荧光原位杂交结合染色体涂抹技术检测骨髓增生异常综合征复杂核型异常

目的探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)及全染色体涂抹(whole chromosome painting,WCP)技术在骨髓增生异常综合征(myelodysplastic syndromes,MDS)复杂核型异常检测中的价值。方法对7例常规R显带具有复杂染色体异常的MDS患者应用M-FISH技术确定复杂染色体的重排及标记染色体的组成,识别微小易位。并进一步采用双色WCP技术验证M-FISH检测的结果。结果M-FISH不仅证实了R显带的结果,而且确定了R带核型分析没有确定的6种标记染色体、9种有不明来源的额外物质增加的染色体、5种衍生染色体的组成和来源及4种被忽略的微小易位。涉及17号染色体的异常及-5/5q-是MDS最为常见的两种染色体异常。WCP技术纠正了一些M-FISH漏检及误检的异常。结论M-FISH是明确复杂染色体异常的很有用的分子生物学工具,WCP是M-FISH技术的重要补充,R带核型分析结合分子细胞遗传学工具M-FISH和WCP可以更加准确地描述复杂染色体异常。     

11例男性染色体异常核型与病因分析

目的应用细胞遗传学检查,对有流产史、发育迟缓的男性患者病因进行分析,探讨对生殖的影响。方法采用外周血染色体G显带技术,对男性患者进行细胞遗传学检测。结果发现异常核型11例,其中染色体易位6例、2例47,XXY、3例大Y染色体。结论男性染色体核型异常是导致流产和发育迟缓的重要因素。     

G显带核型联合染色体微阵列分析在超声异常胎儿产前诊断中的应用进展

G显带核型分析是染色体产前诊断的金标准,然而其常受到细胞培养成功率、细胞分裂指数及标本质量的影响,且不利于快速诊断,也不能检出染色体片段小于5 Mb的微小缺失及微重复。染色体微阵列分析(chromosomal microarray analysis,CMA)能够检出100kb以上染色体微小缺失及微重复,能在一定程度上弥补G显带核型分析的不足。合理地将CMA结合核型分析应用于超声异常胎儿产前诊断,可以提高染色体异常检出率,减少出生缺陷。本文就G显带核型联合CMA在超声异常胎儿产前诊断中的应用进展作一综述。     

Simultaneous detection of centromere-specific probes and chromosome painting libraries by a combination of primedin situ labelling and chromosome painting (PRINS-painting)

In situ techniques for the detection of specific chromosomes using centromeric probes and the decoration of entire chromosomes using chromosome painting are well established. However, in the deciphering of complex chromosomal aberrations it is valuable to be able to detect the centromere and the entire DNA of a specific chromosome in different colours simultaneously on the same metaphase. In this report we describe a combination of the primedin situ labelling (PRINS) technique and chromosome painting for simultaneous visualization of centromere-specific oligonucleotides and chromosome painting libraries. A key feature is that the denaturation step in the PRINS reaction is sufficient to keep the chromosomes denatured for chromosome painting. This means that PRINS and consecutive chromosome painting can be performed as a single procedure (PRINS-painting)     

A chromosome painting method for human sperm chromosomes using fluorescentin situ hybridization

Summary A method of chromosome painting on human sperm chromosomes using fluorescentin situ hybridization (FISH) is introduced. Sperm chromosome slides were prepared afterin vitro fertilization of hamster eggs with human spermatozoa. The slides were treated by RNase A before FISH. Chromosome 4 was clearly and specifically painted in a majority of sperm-derived metaphase plates after an application of whole chromosome painting DNA probes of this chromosome. This is the first report of successful painting on human sperm chromosomes.     

Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation

A novel technique using the incorporation of 5-ethynyl-2''-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.     

A new chromosome banding technique,spectral color banding (SCAN), for full characterization of chromosomal abnormalities

We have developed spectral color banding (SCAN) as a new chromosome banding technique based on spectral analysis of differentially labeled chromosome band-specific painting probes. In this study, we succeeded in displaying a multicolor banding pattern for chromosome 3, which was almost identical to the pattern obtained with the corresponding G-banding. We applied this method to metaphase cells from different normal male donors with various levels of G-banding resolution, ranging from 250 to 850 bands per haploid set. The same multicolor banding pattern was observed in all samples regardless of the length of the chromosomes or the quality of the G-banding. We then used SCAN in a diffuse large B-cell lymphoma case for a complete analysis of the intrachromosomal change for chromosome 3, which could not be fully characterized by G-banding or even by spectral karyotyping (SKY). SCAN could detect the duplicated segment and identify the origin of the chromosome band on the basis of the specific spectral color of each band. This study demonstrates that SCAN is a useful tool for full characterization of chromosomal abnormalities not identified by SKY.     

FISH技术在一例45,X/46,X,i（Xq）Turner综合征中的研究

目的应用荧光原位杂交技术（fluorescence in situ hybridization,FISH）分析一例45,X/46,X,i（Xq）嵌合体,并探讨其形成机理,临床表型与染色体核型的关系。方法通过染色体常规G显带技术,并联合FISH技术,选用X染色体着丝粒特异DNA探针（CSPX）和X染色体长臂全涂抹探针（Xq）,进一步确认异常染色体的来源。结果 G显带分析该患者染色体核型为45,X/46,X,i（Xq）,FISH技术证实了该异常染色体为Xq等臂染色体。结论 X短臂单体长臂三体型Turner综合征患者的临床表型与其染色体核型相关;在常规G显带的基础上,应用FISH技术可准确识别异常染色体,对明确诊断及后续治疗有指导意义。     

Marker chromosome identification by micro-FISH

Micro-FISH was used to elucidate the chromosomal origin of marker chromosomes in three patients. Ten copies of marker chromosomes were collected with microneedles from GTG banded metaphases, transferred to a collecting drop and amplified by means of DOP-PCR. The PCR products were labeled with biotin-14-dATP and used as FISH probes for hybridization to normal metaphase chromosomes and to metaphase chromosomes of the patients (reverse painting). With the generation of chromosome region-specific painting probes by PCR amplification of microdissected DNA and subsequent FISH it was possible to identify the marker chromosomes in all patients. One marker appeared to be derived from the centromere region of the X-chromosome and the proximal third of the long arm, one from the centromere region of chromosome 17 and one marker chromosome was identified as an isochromosome 18p.     

Highly comprehensive karyotype analysis by a combination of spectral karyotyping (SKY), microdissection, and reverse painting (SKY-MD)

A technique disclosing most information about chromosome modifications is the technique of choice for the analysis of chromosome alterations. The newly developed method for microdissection of fluorescence-labeled chromosomes (FISH-MD) can improve upon this expectation in combination with 24-color spectral karyotyping (SKY). The highly efficient way to detect chromosome modifications by SKY and the detailed specification of aberrant chromosomes by FISH-MD prompted us to use both techniques in a combined approach called SKY-MD. First, an overview of chromosomal aberrations is obtained by spectral karyotyping and subsequently the derivative chromosomes recognized are characterized in a highly specific manner by microdissection and reverse painting. A small quantity of isolated material dissected directly from a 24-color metaphase is sufficient to obtain very detailed information about the chromosome regions and the breakpoints involved in the derivative chromosomes. Therefore, the combination of spectral karyotyping and microdissection in one procedure, and reverse painting can characterize chromosomal aberrations with a degree of specificity hitherto unknown from individual karyotyping experiments. In this article we compare the efficiency of both the SKY technique and that of classical microdissection with the efficiency obtained by SKY-MD.     

分子细胞遗传学技术在确诊9p三体综合征中的应用

目的　用分子细胞遗传学技术鉴别常规细胞遗传学难以确定的染色体异常。方法　用染色体涂染、比较基因组杂交(com parative genom ic hybridization,CGH)和多色显带分析技术(colorbandingchrom osom e analysis,RxFISH),对一例G 显带提示为9 号染色体结构异常的病例进行研究。结果　患儿核型为46,XX,9p ,染色体涂染显示两条9 号染色体(包括短臂额外片段)被均匀涂染,CGH 和RxFISH证实为9 号短臂完全重复,核型准确描述为46,XX,dup 9p(p11→p24∷p24→qter)。结论　这些技术手段可以鉴别常规细胞遗传学难以进行诊断的、复杂的染色体结构异常,在基础及临床医学研究领域中,有着重要的应用前景