淋巴瘤比较基因组杂交研究

目的评价比较基因组杂交(CGH)技术在淋巴瘤研究中的应用价值。方法采用CGH技术检测20例淋巴瘤患者核型异常,部分病例与核型分析比较。结果20例淋巴瘤患者CGH核型异常检出率为60%。8例进行常规核型分析的患者,CGH检测出4例常规核型分析未发现的异常。8例弥漫大B细胞淋巴瘤5例发现染色体拷贝数改变,其中3例发现13号染色体长臂扩增。5例滤泡性淋巴瘤中3例分别检测出7q11-21和15q11-14扩增和18号三体。4例小淋巴细胞淋巴瘤中1例6q16-25缺失,1例发现复杂核型异常:1p21-23扩增伴有2p12-ter,9p13-ter,10p13-ter缺失。结论淋巴瘤患者存在多种染色体异常,CGH是一有用的分析淋巴瘤核型异常的分子细胞遗传学工具。     

淋巴瘤比较基因组杂交研究

目的评价比较基因组杂交（CGH）技术在淋巴瘤研究中的应用价值。方法采用CGH技术检测20例淋巴瘤患者核型异常，部分病例与核型分析比较。结果20例淋巴瘤患者CGH核型异常检出率为60％。8例进行常规核型分析的患者，CGH检测出4例常规核型分析未发现的异常。8例弥漫大B细胞淋巴瘤5例发现染色体拷贝数改变，其中3例发现13号染色体长臂扩增。5例滤泡性淋巴瘤中3例分别检测出7q11-21和15c11-14扩增和18号三体。4例小淋巴细胞淋巴瘤中1例6q16—25缺失，1例发现复杂核型异常：1p21—23扩增伴有2p12-ter，9p13-ter，10p13-ter缺失。结论淋巴瘤患者存在多种染色体异常，CGH是一有用的分析淋巴瘤核型异常的分子细胞遗传学工具.     

基于基因芯片的比较基因组杂交方法在分析非特指型外周T细胞淋巴瘤染色体改变中的价值评估

【摘要】目的探讨基于基因芯片的比较基因组杂交（array—basedcomparativegenomichybridiza—tion。Array—CGH）方法检测非特指型外周T细胞淋巴瘤（peripheralT—celllymphoma—nototherwisespeci—fled,PTCL—NOS）的分子遗传学改变特征的临床价值。方法收集2001年10月至2008年12月PTCL—NOS患者组织标本31例,采用1MbArray—CGH检测其基因变化,并用TilepathArray—CGH对检测结果进行验证。数据采用SPSS14．0统计软件进行分析。结果31例PTCL—NOS标本中,有17例（54．8％）出现不同程度的染色体改变,且有基因改变的患者生存期明显短于无基因改变的患者,差异均有统计学意义（P均〈0．05）。经TilepathArray—CGH检测验证,1MbArray—CGH检测的基因改变与TilepathAr—ray—CGH检测的基因改变完全一致。结论Array—CGH可以全面快速检测与肿瘤相关的染色体改变,对研究淋巴瘤特异性遗传特征具有重要意义。     

比较基因组杂交在肿瘤病理诊断中的应用

比较基因组杂交（comparativegenomehybriclization，CGH）是一种将原位荧光杂交技术与数字图象分析相结合，用于检测肿瘤组织DNA异常（缺失、扩增、复制），并在染色体上定位的细胞遗传学新方法（门。与传统分子水平的DNA检测手段相比，在一次实验中即可对整个基因组进行分析，并能确定肿瘤DNA异常发生于哪条染色体，位于染色体的哪个区带，同时还可获得相应的癌基因扩增或抑癌基因丢失的信息。目前这一方法在国外已被用于肿瘤遗传学研究的多个领域，其在肿瘤病理诊断中的应用对阐明肿瘤的发病机制、疾病分类、推断之发展的不同阶段，…     

软组织肿瘤的比较基因组杂交研究进展

比较基因组杂交(comparative genomic hybridization,CGH)是1992年建立起来的重大分子细胞遗传学分析技术,它在对整个基因组DNA拷贝数变异的检测方面是一种有效的方法。通过CGH检测,可找出染色体DNA拷贝数的变异特点,即基因拷贝数的扩增或丢失,从而确定相关的癌基因和抑癌基因所在的区域,为探讨肿瘤的发病机制提供依据。在过去的十几年中,用CGH对各种软组织肿瘤进行研究,已探测到了各种各样的有不同程度特异性的基因组的不平衡性,不仅开辟了探测各种癌症相关基因的新途径,并且找到了一些与临床相关的基因改变,可用于肿瘤的发生、发展、鉴别诊断、预后以及治疗等研究。     

应用荧光原位杂交技术检测慢性淋巴细胞白血病的基因组异常

目的 探讨应用组合荧光原位杂交(panel fluorescence in situ hybridization,panel FISH)技术对慢性淋巴细胞白血病(chronic lymphocytic leukaemia,CLL)基因组异常检测的价值.方法 分别应用序列探针D13S25(13q14.3)、RB1、p53、ATM(11q23)和着丝粒探针12号(CSP12)等5种荧光素标记的DNA探针,对17例CLL患者进行FISH检测,并和常规细胞遗传学检测结果进行比较.结果 17例CLL患者中,常规细胞遗传学检测出1例(1/17)有染色体异常,为49,XX,+3,+8,+18;组合FISH检测出10例(10/17)有染色体异常,包括D13S25缺失4例、ATM缺失2例、p53缺失1例、D13S25合并RB1同时缺失2例、多种异常1例.FISH检测的总检出率高于常规细胞遗传学检测.结论 组合FISH技术是检测CLL患者染色体基因组异常的有效手段,与常规细胞遗传学方法相结合则可明显提高CLL染色体异常的检出率.     

比较基因组杂交技术在软组织肉瘤诊断中的应用

比较基因组杂交(CGH)技术是在荧光原位杂交(FISH)基础上，结合消减技术发展起来的一种能快速、全面分析染色体基因组获得和缺失的分子遗传学方法。它不需要特殊探针，能进行全基因组检测，并可在正常中期染色体上定位。目前CGH技术是对整个染色体扫描分析最常用的技术，在软组织肉瘤的诊断和研究中有重要意义。     

增生性瘢痕与瘢痕疙瘩DNA拷贝数变化的差异分析

背景:近年来临床遗传学和分子生物学研究均表明,瘢痕疙瘩的形成与遗传具有密切的关系.但增生性瘢痕与遗传是否有关,目前尚未明确.目的:了解增生性瘢痕与瘢痕疙瘩在遗传学改变上的异同.设计、时间及地点;对比观察,实验于2007-03/2008-12在广东医学院完成.材料:瘢痕标本均来自2003-01/2008-12广东医学院附属医院整形外科门诊及住院患者16例,其中增生性瘢痕10例,男3例,女7例,年龄20～50岁;瘢痕疙瘩6例,男1例,女5例,年龄19～46岁.方法:提取瘢痕疙瘩及增生性瘢痕组织DNA,应用比较基因组杂交技术观察增生性瘢痕及瘢痕疙瘩基因组的不平衡即遗传物质的丢失或扩增情况,比较两者间DNA拷贝数变化的差异.主要观察指标:①两组DNA拷贝数的缺失率的比较.②两组DNA拷贝数的扩增率的比较.结果:增生性瘢痕组未发现特异区域的DNA拷贝数的高频率缺失或扩增;瘢痕疙瘩组出现高频率的DNA拷贝数缺失的染色体是1,16,20号及22号染色体,未发现特异区域的DNA拷贝数的高频率扩增.两组1,16,20,22染色体DNA拷贝数的缺失率相比较,瘢痕疙瘩组明显高于增生性瘢痕组(P<0.05).结论:与瘢痕疙瘩相比,增生性瘢痕不存在明显的DNA拷贝数缺失或扩增,增生性瘢痕的形成与发展可能与遗传没有直接的关系.     

array-CGH在产前诊断染色体疾病中的应用

摘要: 微阵列比较基因组杂交技术（array-based comparative genomic hybridization, array-CGH）结合了比较基因组杂交技术（CGH）、微阵列芯片技术（micro-array）的优势,在分子遗传学中广泛应用于全基因组水平的拷贝数分析。array-CGH 在产前诊断染色体疾病中的应用相比于传统的细胞遗传学核型分析技术以及荧光原位杂交技术（FISH）、多重荧光定量PCR（QF-PCR）、多重连接探针扩增技术（MLPA）等分子遗传学技术具有高通量、高分辨、高灵敏度、操作自动化等优势;能够检测出相当一部分常规核型分析技术不易发现的染色体微缺失和微重复综合征,以及亚端粒或者其他不平衡的染色体重排。目前用array-CGH进行全基因组扫描进行产前诊断,判断和评估所检出的拷贝数变异（CNVs）的临床意义有一定难度。对不同位点CNVs出现频率及临床意义研究可能会是近期研究热点之一。     

NGS和MLPA技术检测2例马凡综合征患者FBN1基因缺失突变

目的对2例马凡综合征(Marfan's syndrome,MFS)患者的原纤维蛋白-1基因(FBN1)进行突变检测。方法提取患者的外周血全基因组DNA,用第二代测序技术(NGS)和多重连接探针扩增技术(MLPA)对FBN1进行突变筛查,对这2种方法提示有拷贝数异常的外显子进行PCR和DNA Sanger测序以证实突变。结果 NGS和MLPA技术检测均显示1例患者有18号外显子缺失突变,另1例患者其56号外显子有缺失突变。经PCR和Sanger测序证实前者18号外显子及其两侧翼区有大片段缺失c.2114-2357_2167+747del3158bp,后者第56号外显子及其内含子有9 bp的缺失突变c.6864_c.6871+1del CTGTGTAGG。结论 NGS和MLPA技术有助于筛查基因组缺失突变,但仍需借助Sanger测序等方法验证。     

Detection of MYCN amplification and chromosome 1p36 loss in neuroblastoma by cDNA microarray comparative genomic hybridization.

BACKGROUND: In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma. AIM: The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer. METHODS: DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH. RESULTS: Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification. DISCUSSION: Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.     

Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors

Identification of prognostic and predictive genomic markers requires long-term clinical follow-up of patients. Extraction of high-quality DNA from archived formalin-fixed, paraffin-embedded material is essential for such studies. Of particular importance is a robust reproducible method of whole genome amplification for small tissue samples. This is especially true for high-resolution analytical approaches because different genomic regions and sequences may amplify differentially. We have tested a number of protocols for DNA amplification for array-based comparative genomic hybridization (CGH), in which relative copy number of the entire genome is measured at 1 to 2 mb resolution. Both random-primed amplification and degenerate oligonucleotide-primed amplification approaches were tested using varying amounts of fresh and paraffin-extracted normal and breast tumor input DNAs. We found that random-primed amplification was clearly superior to degenerate oligonucleotide-primed amplification for array-based CGH. The best quality and reproducibility strongly depended on accurate determination of the amount of input DNA using a quantitative polymerase chain reaction-based method. Reproducible and high-quality results were attained using 50 ng of input DNA, and some samples yielded quality results with as little as 5 ng input DNA. We conclude that random-primed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples.     

不同免疫表型弥漫大B细胞淋巴瘤的抑癌基因TP53缺失分析

目的探讨不同免疫表型的弥漫大B细胞淋巴瘤(diffuse large B-cell 1ymphoma,DLBCL)抑癌基因TP53缺失情况的差异。方法根据淋巴瘤细胞免疫标志物CD10、Bcl-6及MUM-1的表达情况,将57例DLBCL石蜡包埋组织标本分为GCB(生发中心样B细胞)与non-GCB(非生发中心样B细胞)淋巴瘤两类,用荧光原位杂交(FISH)技术检测GCB与non-GCB类中TP53缺失情况。结果 24例GCB类中有4例(16.7%)TP53缺失,33例non-GCB类中有14例(42.4%)TP53缺失。二者TP53缺失率差异有统计学意义(P<0.05)。结论 non-GCB类患者TP53缺失发生率较高,TP53缺失是non-GCB类预后差的可能原因之一。FISH可以快速、准确、灵敏地检测出TP53的缺失。     

Utility of oligonucleotide array-based comparative genomic hybridization for detection of target gene deletions

BACKGROUND: direct DNA sequencing is the primary clinical technique for identifying mutations in human disease, but sequencing often does not detect intragenic or whole-gene deletions. Oligonucleotide array-based comparative genomic hybridization (CGH) is currently in clinical use to detect major changes in chromosomal copy number. METHODS: a custom oligonucleotide-based microarray was constructed to provide high-density coverage of an initial set of 130 nuclear genes involved in the pathogenesis of metabolic and mitochondrial disorders. Standard array CGH procedures were used to test patient DNA samples for regions of copy number change. Sequencing of regions of predicted breakpoints in genomic DNA and PCR analysis were used to confirm oligonucleotide array CGH data. RESULTS: oligonucleotide array CGH identified intragenic exonic deletions in 2 cases: a heterozygous single-exon deletion of 4.5 kb in the SLC25A13 gene [solute carrier family 25, member 13 (citrin)] in an individual with citrin deficiency and a homozygous 10.5-kb deletion of exons 13-17 in the ABCB11 gene [PFIC2, ATP-binding cassette, sub-family B (MDR/TAP), member 11] in a patient with progressive familial intrahepatic cholestasis. In 2 females with OTC deficiency, we also found 2 large heterozygous deletions of approximately 7.4 Mb and 9 Mb on the short arm of the X chromosome extending from sequences telomeric to the DMD gene [dystrophin (muscular dystrophy, Duchenne and Becker types)] to sequences within or centromeric to the OTC gene (ornithine carbamoyltransferase). CONCLUSIONS: these examples illustrate the successful use of custom oligonucleotide arrays to detect either whole-gene deletions or intragenic exonic deletions. This technology may be particularly useful as a complementary diagnostic test in the context of a recessive disease when only one mutant allele is found by sequencing.     

FICTION技术在检测多发性骨髓瘤遗传学异常中的应用

目的 探讨联合免疫荧光和荧光原位杂交(FISH)的FICTION技术在多发性骨髓瘤(MM)遗传学异常检测中的应用价值.方法 采集18例MM患者和2例浆细胞白血病患者的骨髓标本,分离单个核细胞制作滴片.从细菌人工染色体文库中选取覆盖IgH、MMSET待测基因位点的质粒,用缺口平移法制备带有半抗原检测标签的核酸探针.在经CD138标记和酪胺信号放大的细胞滴片标本上,使用上述自制探针[t(4;14)、t(11;14)和t(14;16)]和商品化直标缺失探针(13q和p53)进行FISH检测.结果 20例患者标本均使用上述5种探针进行检测,其中检出t(4;14)4例,t(11;14)6例,t(14;16)1例,p53缺失3例,13q缺失8例;另有4例未检测出此5种异常.结论 应用FICTION技术原位分析骨髓中特定瘤细胞亚群的特征性遗传学异常,能够提高FISH检测的效率和敏感性,并可作为对MM患者遗传学分层诊断的初筛实验,指导治疗并判断预后.     

FICTION技术在检测多发性骨髓瘤遗传学异常中的应用

目的 探讨联合免疫荧光和荧光原位杂交(FISH)的FICTION技术在多发性骨髓瘤(MM)遗传学异常检测中的应用价值.方法 采集18例MM患者和2例浆细胞白血病患者的骨髓标本,分离单个核细胞制作滴片.从细菌人工染色体文库中选取覆盖IgH、MMSET待测基因位点的质粒,用缺口平移法制备带有半抗原检测标签的核酸探针.在经CD138标记和酪胺信号放大的细胞滴片标本上,使用上述自制探针[t(4;14)、t(11;14)和t(14;16)]和商品化直标缺失探针(13q和p53)进行FISH检测.结果 20例患者标本均使用上述5种探针进行检测,其中检出t(4;14)4例,t(11;14)6例,t(14;16)1例,p53缺失3例,13q缺失8例;另有4例未检测出此5种异常.结论 应用FICTION技术原位分析骨髓中特定瘤细胞亚群的特征性遗传学异常,能够提高FISH检测的效率和敏感性,并可作为对MM患者遗传学分层诊断的初筛实验,指导治疗并判断预后.

Abstract：

Objective To investigate the diagnostic value of FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms) technique, combining immunofluorescence and fluorescence in situ hybridization (FISH), to detect genetic aberrations in multiple myeloma (MM). Methods Bone marrow samples were collected from 18 MM and 2 plasma cell leukemia (PCL)patients. Probes targeting IgH and MMSET were prepared using a Nick Translation Kit from Bacterial artificial chromosome (BAC) clones. The immunophenotyping was achieved via the CD138 tyramide signal amplification (TSA)-mediated immunofluorescence, followed by FISH with the prepared probes [t (4;14), t (11;14), t(14;16)] and the commercial deletion probes (13q and p53) to detect common genetic aberrations in MM. Results All the 20 samples were assayed with the probes mentioned above, and revealed 4 cases with t(4;14) ,6 with t(11 ;14), 1 with t(14;16), 3 with p53 deletion; and 8 with 13q deletion. The remaining 4 cases had none of the 5 aberrations. Conclusion FICTION technique facilitates the detection of genetic abnormalities of MM in situ; enhances both efficiency and sensitivity of positive det~tion, thus, could be used as the screening test of molecular diagnosis of MM to guide coming-up risk-adapted therapy and evaluate prognosis.     

FICTION技术在检测多发性骨髓瘤遗传学异常中的应用

目的 探讨联合免疫荧光和荧光原位杂交(FISH)的FICTION技术在多发性骨髓瘤(MM)遗传学异常检测中的应用价值.方法 采集18例MM患者和2例浆细胞白血病患者的骨髓标本,分离单个核细胞制作滴片.从细菌人工染色体文库中选取覆盖IgH、MMSET待测基因位点的质粒,用缺口平移法制备带有半抗原检测标签的核酸探针.在经CD138标记和酪胺信号放大的细胞滴片标本上,使用上述自制探针[t(4;14)、t(11;14)和t(14;16)]和商品化直标缺失探针(13q和p53)进行FISH检测.结果 20例患者标本均使用上述5种探针进行检测,其中检出t(4;14)4例,t(11;14)6例,t(14;16)1例,p53缺失3例,13q缺失8例;另有4例未检测出此5种异常.结论 应用FICTION技术原位分析骨髓中特定瘤细胞亚群的特征性遗传学异常,能够提高FISH检测的效率和敏感性,并可作为对MM患者遗传学分层诊断的初筛实验,指导治疗并判断预后.