用染色体涂染技术分析五例染色体结构异常

目的 结合G－显带核型分析诊断染色体易位,并对G显带技术难以鉴定的微小易位进行分析。方法 采用生物素标记的显微切割备的X、Y、14q,10号染色体特异性探针,与患者外周血培养淋巴细胞中期染色体进行荧光原位杂交。结果 室温存放近10年的标本,－80℃冻存标本及新鲜标本均可看到清晰的杂交信号,染色体结构异常很清楚。结论 染色体涂染技术结合G显带核型分析,可以准确识别G显带技术难以鉴定的染色体微小易位。     

伴t(1;7)易位骨髓增生异常综合征的双色荧光原位杂交研究

目的 通过对5例伴有t（1；7）易位的骨髓增生异常综合征（myelodysplastic syndromes,MDS）患者进行研究，并进一步确定易位杂色体着丝粒的组成和来源。方法 采用骨髓细胞直接法或短期培养法制备染色体，应用R显带技术进行核型分析，应用荧光素SpectrumRed标记的1号染色体着丝粒特异性α卫生DNA探针和荧光素SpectrumGreen标记的7号染色体着丝粒特异性α卫星DNA探针，对其中3例患者进行双色荧光原位杂交（fluorescence in situ hybridization,FISH）研究。结果 5例患者均有t(1;7)易位。双色FISH显示其中3例患者t（1;7) 位所致衍生染色体的着丝粒均由红绿两个信号融合而成。结论 双色FISH证实MDS患者t(1;7）易位染色体着丝粒由1号和7号染色体着丝粒共同组成。     

光谱核型分析技术在染色体异常诊断中的应用

目的建立稳定的光谱核型分析技术,并评价其在染色体异常诊断中的应用价值。方法应用光谱核型分析技术对16例外周血和1例羊水细胞染色体样本进行诊断,并与G-显带诊断结果进行比较。结果所建立的光谱核型分析技术不但成功地对全部15例G-显带诊断结果为正常或平衡易位的样本进行了正确诊断,而且对G-显带无法识别的1例外周血样本的衍生染色体片段和1例羊水样本的标记染色体亦明确了来源。结论光谱核型分析技术对染色体异常的诊断具有很高的敏感性和特异性,并且在明确传统核型分析技术不能分析的标记染色体或衍生染色体片段的来源方面具有重要应用价值。     

荧光原位杂交技术在复杂染色体异常产前诊断中的应用

目的 探讨荧光原位杂交技术(fluorescence in situ hybridization,FISH)在复杂染色体异常产前诊断中的应用价值.方法 对8例羊水、3例脐血常规G显带具有复杂染色体异常的产前诊断孕妇,应用FISH技术确定其复杂染色体重排及标记染色体的组成.结果 FISH技术证实了G-显带平衡易位的结果,同时明确了3例羊水中衍生染色体的组成、2例脐血中标记染色体的来源.结论 FISH技术具很高的敏感性和特异性,是明确染色体异常重要的分子细胞遗传学工具,其在产前诊断中的应用,可为临床提供更准确全面的实验依据.     

骨髓增生异常综合征的临床与细胞遗传学研究

目的研究骨髓增生异常综合征（myelodysplastic syndrome，MDS）的细胞遗传学、血液学与预后的相互关系。方法采用骨髓直接法和24小时短期培养法制备染色体标本，用R显带技术，对50例MDS进行核型分析。结果50例MDS中，发现有异常核型22例，发生率44．0％（22／50）。异常类型6种：2例add（8）；4例-7；4例5q-；9例7q-；2例20q-；1例6q-。结论5q-，-7，7q-是MDS中最为常见的染色体核型异常，伴有5q-染色体核型异常的预后较好，而伴有-7，7q-核型异常的预后不良。细胞遗传学在MDS的诊断、病情发展和预后判断中有着至关重要的作用。     

多重荧光原位杂交技术体系的建立及其在检测白血病复杂核型异常中的应用

目的：建立多重荧光原位杂交技术（multiplex fluorescence in situ hybridization，M-FISH）体系，探讨其在检测白血病复杂核型异常中的应用。方法：联合应用常规核型分析、染色体涂抹（chromosome painting，CP）、染色体荧光原位杂交（fluorescence in situ hybridization，FISH）和M-FISH方法分析了两例白血病复杂核型。结果：对其中1例常规核型分析显示为46，XY，der(9)t(9；12)的急性淋巴细胞白血病-L2型病例，M-FISH检出其具有复杂的染色体易位；46；XY，der（2）t(2；9)，der(9)t(9；12；22)。对另1例常规核型难以分析的急性单核细胞白血病-M5型病例，M-FISH检出其复杂核型是46，XY，der(2)t(2；17)，der(10)t(10；11；17)，der(11)t(11；？)。进下不用染色体涂抹和双色FISH证实了M-FISH的结果，并发现MLL基因（mixed lineage leukemia gene）受累。结论：M-FISH是筛选白血病复杂染色体异常的理想方法，对阐明所有白血病、乃至其它肿瘤性疾病和遗传性疾病的染色体易位和基因改变有广阔的应用前景。     

一例伴有t(1;18)(p31;p11)易位的骨髓增生异常综合征

目的报告一例伴有t(1；18)(p31；p11)的骨髓增生异常综合征(myelodysplastic syndrome，MDS)。方法骨髓细胞24h培养后按常规方法制备染色体，采用R显带技术进行染色体核型分析；以1号和18号整条染色体涂染探针对其进行染色体涂染检测。结果常规细胞遗传学方法和染色体涂染分析均证实该患者具有t(1；18)(p31；p11)克隆性染色体异常。结论t(1；18)(p31；p11)易位是一种罕见的再现性染色体核型异常，在MDS中属于首次报道。     

自贡地区630例智力低下、发育异常患者的细胞遗传学分析

目的通过对智力低下、发育异常患者进行细胞染色体核型分析,探讨智力低下、发育异常与染色体异常的关系。方法对630例智力低下、发育异常患者外周血进行淋巴细胞培养,制备染色体进行G显带,分析其染色体核型。结果630例智力低下、发育异常患者中染色体异常者219例,栓出率为34．8％,包括染色体数目异常、嵌合体、易位、倒位、及染色体多态性等多种改变,分别占20．5％、5．9％、4．1％、3．3％及1．0％,其中21-三体综合征核型最多见,共138例,占异常核型的63．O％,包括标准型、易位型及嵌合体型,分别占21-三体核型中的74．6％、15．2％及10．1％。结论染色体异常与智力低下、发育异常密切相关,应引起高度重视,对高危人群进行染色体检查,对临床诊断、治疗及指导优生优育具有重要意义。     

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.     

组合探针荧光原位杂交检测骨髓增生异常综合征常见染色体异常

目的：评价组合探针荧光原位杂交（fluorescence in site hybridization,FISH)在检测骨髓增生异常综合征（myelodysplastic syndrome,MDS）常见染色体异常中的价值。方法：应用YAC248F5（5q31)、YAC938G5（7q32）、CEP8、YAC912C3（20q12）4种DNA探针，对核型未知的20例MDS患者进行FISH检测－5／5q-、－7／7q-、＋8、20q－等常见染色体异常，并与常规细胞遗传学分析结果相比较。结果：20例MDS患者中，组合探针FISH检出13例有常见染色体异常（其中5例＋8，1例－5／5q-，5例20q-,1例5q-合并20q-，复杂异常1例）；而常规细胞遗传学发现5例常见染色体异常，1例＋21，复杂异常1例，标记染色体1例，正常5例。结论：组合探针FISH是筛查MDS患者常见染色体异常的有效手段。     

荧光原位杂交技术检测多发性骨髓瘤复杂核型异常

目的探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)技术联合荧光原位杂交(fluorescence in situ hybridization,FISH)技术在检测多发性骨髓瘤(multiplemyeloma,MM)染色体异常中的应用价值及13q14缺失、IgH相关易位和17p13缺失的发生率。方法联合应用常规细胞遗传学(conventional cytogenetics,CC)方法及M-FISH和一组包括13q14(D13S319),14q32(IgH基因)和17p13(p53基因)探针的FISH技术分析了7例伴有复杂染色体异常的MM患者骨髓标本。结果M-FISH明确了CC分析中没有明确的异常,共检出12种染色体数目异常和29种结构异常,其中,1号染色体异常、13号染色体缺失和与14q32相关的易位最为多见。FISH检出6例伴有13q14缺失;4例伴有17p13缺失;5例伴有一个14q32相关易位,两例还伴有涉及两个14q32的易位。结论M-FISH联合FISH技术可以明确CC分析中复杂染色体异常,并发现和纠正CC分析中漏检及误检的异常,为MM染色体异常的研究提供了一种理想的     

Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases

Chromosomal abnormalities are found by conventional cytogenetic (CC) analysis in about 50% of myelodysplastic syndromes (MDS) and 70% of acute myeloid leukemias (AML). When cytogenetic abnormalities are complex, multiplex fluorescence in situ hybridization (M-FISH) can help clarify complex chromosomal abnormalities and identify rearrangements with prognostic value or cryptic translocations, which could be preliminary steps in identifying new genes. We studied by M-FISH 28 cases of MDS and AML with complex chromosomal abnormalities, 10 of them were therapy-related. M-FISH allowed the characterization of unidentified chromosomal material in 26 cases (93%). One or several unbalanced rearrangements were observed in 27 cases (96%), generally interpreted as deletions or additional material by CC. Among those translocations, 4 involved 3 chromosomes. Eighteen cryptic translocations undetected by CC were found in 13 cases. By FISH analysis using locus specific probes, TP53 deletion, additional copies of MLL, and additional copies or deletions of RUNX1/AML1 were observed in 16, 4, and 3 cases, respectively. Thus, M-FISH is an important tool to characterize complex chromosomal abnormalities which identified unbalanced and cryptic translocations in 96% and 46% of the cases studied, respectively. Complementary FISH helped us identify involvement of TP53, MLL, and RUNX1/AML1 genes in 82% of cases, confirming their probable role in leukemogenesis.     

多重荧光原位杂交技术检测多发性骨髓瘤复杂核型异常

目的 探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)技术在多发性骨髓瘤(multiple myeloma,MM)复杂核型异常(complex chromosomal aberrations,CCAs)检测中的价值.方法 对10例常规细胞遗传学(conventional cytogenetics,CC)方法检测具有复杂核型的MM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.结果 M-FISH证实了CC显示的29种结构异常,并进一步明确了1p-、6q-、9q-、9q+、+8q+×2、14q+、?14q、der(4)、der(22)、der(1)t(1;?)(q10;?)、der(3)、del(7)的具体来源;同时也发现CC分析没有发现或不能识别的21种异常,其中t(2;15)(q33;q22)、t(6;7)(q23;q34)、t(8;11)(q24;q23)、t(1;14)(q10;q32)和t(X;1)(q26;q25)是新发现的核型异常.这10例随访的MM患者病例资料中9例已死亡,中位生存期仅为23个月,较公认的MM患者的平均生存期34个月明显缩短.结论 对伴有CCAs的MM患者,M-FISH技术可以明确CC分析中复杂染色体异常,并发现和纠正CC分析中漏检及误检的异常,为MM的染色体异常研究提供了一种重要的方法,已经成为精确染色体核型分析所不可缺少的手段之一.     

Identification of cytogenetic subclasses and recurring chromosomal aberrations in AML and MDS with complex karyotypes using M-FISH.

Complex chromosomal aberrations (CCAs) can be detected in a substantial proportion of AML and MDS patients, de novo as well as secondary or therapy-related, and are associated with an adverse prognosis. Comprehensive analysis of the chromosomal rearrangements in these complex karyotypes has been hampered by the limitations of conventional cytogenetics. As a result, our knowledge concerning the cytogenetics of these malignancies is sparse. Here we describe a multiplex-FISH (M-FISH) study of CCAs in 36 patients with AML and MDS. M-FISH generated a genome-wide analysis of chromosomal aberrations in CCAs, establishing several cytogenetic subgroups. -5/5q- was demonstrated in the majority of patients (86%). Other rearrangements (present with or without -5/5q-) included: deletion of 7q (47%), 3q rearrangements (19%), and MLL copy gain or amplification (17%). These genetic subgroups seem to display biological heterogeneity: MLL copy gain or amplification in association with 5q- was detected only in AML patients and was significantly associated with extremely short survival (median overall survival: 30 days, P = 0.0102). A partially cryptic t(4;5)(q31;q31), a balanced t(1;8)(p31;q22), and an unbalanced der(7)t(7;14)(q21;q13) were detected as possible new recurrent rearrangements in association with CCAs. Novel reciprocal translocations included t(5;11)(q33;p15)del(5)(q13q31) and t(3;6)(q26;q25). We conclude that AML and MDS with CCAs can be subdivided into molecular cytogenetic subclasses, which could reflect different clinical behavior and prognosis, and that three recurrent chromosomal aberrations are associated with karyotype complexity.     

Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding

We report the use of multiplex fluorescence in situ hybridization (M-FISH) to resolve chromosomal aberrations in seven established melanoma cell lines with hypotriploid to hypertetraploid complex karyotypes. By simultaneous identification of all human chromosomes in single FISH experiments using a set of 52 directly labeled, whole chromosome painting probes, cryptic chromosomal translocations and the origin of unclear chromosomal material in structural rearranged and marker chromosomes could be identified, refining the tumor karyotypes in all seven cell lines. The number of structural aberrations in each cell line assigned with combined M-FISH and DAPI banding analysis ranged from 15 to 45. Altogether, 275 breakpoints could be assigned to defined chromosomal regions or bands. The chromosome arms 1p, 6q, 7p, 9p, and 11q which are known to be nonrandomly associated with melanoma tumorigenesis, were frequently involved in chromosomal breaks and/or copy number changes. This study also demonstrated the practical usefulness of combining M-FISH with conventional cytogenetic banding techniques for the characterization of complex tumor karyotypes with massive genomic alterations.     

伴复杂核型异常的髓系恶性血液病中17号染色体异常分析

目的 研究伴复杂核型异常(complex chromosomal abnormalities,CCAs)的髓系恶性血液病中17号染色体的异常特征.方法 经R显带常规细胞遗传学分析显示CCAs的73例髓系恶性血液病,包括21例急性髓系白血病(acute myeloid leukemia,AML)、36例慢性髓系白血病(chronic myeloid leukemia,CML)、16例骨髓增生异常综合征(myelodysplastic syndrome,MDS),并进一步多重荧光原位杂交分析.结果 73例伴CCAs的髓系恶性血液病中,17号染色体异常最常见,占46.5%(34/73),其中AML12例,CML13例,MDS9例,9例CML慢性期患者均未见17号染色体异常.结构异常较多见,总发生率为43.8%(32/73);AML、CML、MDS3组发生率分别为52.4%(11/21)、33.3%(12/36)、56.3%(9/16);所有病例中发生数目异常共15.1%(11/73),三组发生率分别为25.0%(3/12)、38.5%(5/13)、33.3%(3/9),11例数目异常均为-17.有9例同时出现数目异常和结构异常.伴有17号染色体的结构异常中,以非平衡易位多见,3组分别为16、15、8个;平衡易位2个,分别为发生于AML中的t(15;17)及发生于CML中的t(15;17;22).17号染色体结构易位的对手染色体多变,包括了除5号、6号和22号外的所有染色体.结构易位频率最高的对手染色体是15号,占8.2%(6/73);其次为2号,占5.4%(4/73).6例存在17号与15号易位的病例中5例为急性早幼粒白血病,1例为CML急变期.结论 伴CCAs的髓系恶性血液病中17号染色体异常发生率高,以结构异常为主.所有的数目异常均为-17;结构异常以非平衡易位多见.     

一例急性早幼粒细胞白血病同时伴有ins(15;17),t(2;17;20)和三体8异常

目的 对1例伴有ins(15;17),t(2;17;20),+8复杂异常的急性早幼粒细胞白血病(acute promyelocytie leukemia,APE)病例进行细胞和分子遗传学研究.方法 按常规制备染色体,以R显带技术进行核型分析,并先后作多色荧光原位杂交(multiplex fluoresence in situ hybridization,M-FISH)、染色体涂染和PML-RARa双色FISH检测.结果 R显带核型分析为47,XY,2q-,+8,17q+,20p+;M-FISH检测为:47,XY,t(2;17;20)(q24;q21;p13),+8;染色体涂染证实了2qZ4以下片段易位到17q21上和17q21以下片段易位到20p13上;双色FISH示17号染色体上RARa(retinoic acid receptora,RARe)基因部分片段插入到15号染色体形成PNL-RARa融合基因,即ins(15;17)(q22;q21.1q21.3).结论 FISH技术是明确隐匿/插入易位的可靠手段,凡形态学拟诊为APL而常规核型分析未发现t(15;17)者均应进行FISH检测.     

Multiplex fluorescence in situ hybridization and cross species color banding of a case of chronic myeloid leukemia in blastic crisis with a complex Philadelphia translocation

Exciting new techniques in molecular cytogenetics--namely, spectral karyotyping, multiplex fluorescence in situ hybridization (M-FISH), and cross species color banding--have been recently developed. An increasing number of reports demonstrate the success of these procedures in providing additional cytogenetic information--identifying marker chromosomes and revealing the presence of previously undetected chromosomal changes. However, these procedures have their limitations, and their absolute sensitivity in the accurate identification of subtle chromosomal abnormalities remains to be established. M-FISH and color banding have been applied to a case of chronic myeloid leukemia with a complex Philadelphia translocation involving chromosomes 9, 17, and 22, which had initially been identified from G-banded chromosome analysis. The abnormalities were confirmed by chromosome "painting" and specific probes. Although M-FISH and color banding revealed no additional cryptic chromosomal changes, this study has clearly demonstrated the success of these multiple color FISH approaches in the accurate characterization of a complex rearrangement with subtle abnormalities.     

Fluorescence in situ hybridization confirmation of 5q deletions in patients with hematological malignancies

Fluorescence in situ hybridization (FISH) using specific probes for the 5q31-32 region and a whole chromosomal painting (WCP) probe for chromosome 5 were used to corroborate the results of classical cytogenetic examinations performed on G-banded chromosomes of 77 patients with hematological malignancies. Using classical cytogenetic methods, we suspected the presence of clones with a deletion 5q in 63 patients, and complex rearrangements with involvement of chromosome 5 in 14 other cases. Fluorescence in situ hybridization proved the occurrence of deletion 5q31 in 23 patients and ascertained translocations of part of the long arms of deleted chromosome 5 with missing region 5q31 in 12 patients. In 2 cases, the 5q31 region was translocated to other chromosomes as a part of complex rearrangements. The combination of classical cytogenetics and FISH with specific probes for the 5q31 band yielded cytogenetic results in 35 cases. Routine FISH detection of deleted regions was possible by commercially available cosmid probes for the 5q31 chromosomal band. The interpretation of small deletions and frequent involvement of the deleted chromosomes 5 in complex translocations were ascertained by WCP probes.     

Molecular cytogenetic analysis of two primary squamous cell carcinomas of the lung using multicolor fluorescence in situ hybridization

To fully characterize the numerous chromosomal aberrations in two human squamous cell carcinomas (SCCs) of the lung, molecular cytogenetic characterization was carried out utilizing conventional banding analysis and multicolor fluorescence in situ hybridization (mFISH), providing simultaneous color discrimination of all 24 human chromosomes. Both tumors displayed complex aneuploid karyotypes with a host of numerical and structural chromosome abnormalities. Structural aberrations common to both SCCs included rearrangements of chromosomes 1, 3p, 7q, and 8q, contributing to net loss of chromosomal sequences on 1p, 3p, and 8p, and a net gain of 8q. The recently introduced mFISH technique enabled the disclosure of cryptic translocations and the chromosomal composition of previously unrecognized marker chromosomes. Furthermore, mFISH greatly enhanced the ability to delineate chromosomal breakpoints when integrating banding information from conventional banding analysis. Eventually, the application of mFISH as a powerful approach to refine complex tumor karyotypes is expected to result in a more detailed and complete picture of cytogenetic events associated with the development and progression of solid tumors.