1例Angleman 综合征合并动脉导管未闭患儿的细胞分子遗传学研究

目的:对1例Angleman综合征合并动脉导管未闭患儿进行细胞分子遗传学研究.方法:用Affymetrix Cytogenetics 2.7M基因芯片、荧光原位杂交(florescence in situ hybridization,FISH)、G-显带、Ag-NOR染色等技术,对1例智力低下合并不语、兴奋、共济运动失...     

Detection of translocations diagnostic for Berkitt's lymphoma by fluorescent in situ hybridization on histological sections of paraffin blocks

AIM: To test feasibility of detection of translocations which are diagnostic for Berkitt's lymphoma with the method of fluorescence in situ hibridization (FISH) on histological sections of paraffin blocks. MATERIAL AND METHODS: FISH on histological sections for detection of t(8;14)(q24;q32) and variant t(2;8)(p12;q24) and t(8;22)(q24;q11) was performed on the material obtained from 53 patients with typical clinical, morphological and immunological picture. DNA probe LSI IgH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe (Vysis, USA), for variant translocations DNA-probe LSI MYC, Dual color, Break Apart Rearrangement Probe (Vysis, USA) were used. RESULTS: Histological material from 31 patients contained translocations characteristic for LB: in 29 (93.5%)--t(8;14)(q24;q32), in 2--variant rearrangements of locus of gene c-myc. Translocation t(8;14)(q24;q32) and its variants were not detected in 22 patients, the diagnosis was changed for diffuse large B-cell lymphoma (DLBCL). CONCLUSION: Typical for BL clinical, morphological and immunological picture may present in extra-nodal diffuse large B-cell lymphoma with high proliferative activity. Differential diagnosis between BL and the latter lymphoma is possible only basing on detection of translocation t(8;14)(q24;q32) or its variants. If it is impossible to obtain native material, FISH on histological sections of parasffin blocks is the only possible method of differential diagnosis.     

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.     

Duplication of intrachromosomal insertion segments 4q32→q35 confirmed by comparative genomic hybridization and fluorescent in situ hybridization

A 35-year-old man with infertility was referred for chromosomal analysis. In routine cytogenetic analysis, the patient was seen to have additional material of unknown origin on the terminal region of the short arm of chromosome 4. To determine the origin of the unknown material, we carried out high-resolution banding, comparative genomic hybridization (CGH), and FISH. CGH showed a gain of signal on the region of 4q32→q35. FISH using whole chromosome painting and subtelomeric region probes for chromosome 4 confirmed the aberrant chromosome as an intrachromosomal insertion duplication of 4q32→q35. Duplication often leads to some phenotypic abnormalities; however, our patient showed an almost normal phenotype except for congenital dysfunction in spermatogenesis.     

Chromosomal abnormalities in non-small cell lung carcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphase fluorescence in situ hybridization

Human lung carcinogenesis is accompanied by complex chromosomal changes that may be detected in interphase cells by fluorescence in situ hybridization (FISH) assay using recently developed multitarget DNA probes. Touch preparations of 20 non-small cell lung carcinomas, sputum specimens from 3 patients with lung cancer and from 11 ex-smokers without lung cancer, and cultured benign bronchial epithelium of 42 high-risk smokers, 9 of whom had concurrent invasive carcinoma, were tested using a four-color FISH probe (LAVysion) targeting centromere 6, 5p15.2, 7p12 (EGFR), and 8q24 (MYC). Significantly high frequencies of abnormal cells were found in each of the 20 NSCLC (100%) and in the 3 sputum specimens from lung cancer patients. None of the cytologically normal sputa contained FISH abnormalities. Cultured bronchial epithelial cells from 11 of 42 patients (26%) were abnormal for at least one probe. Abnormal FISH patterns had no association with gender, presence of tumor or histology. Multicolor FISH can readily detect chromosomal abnormalities in imprints and sputa from lung carcinomas. Chromosomal aneusomy is also frequent in bronchial epithelial cells from long-term smokers. The prognostic significance of the multicolor LAVysion FISH probe set should be validated in a controlled clinical trial.     

荧光原位杂交技术快速诊断胎儿常见染色体数目异常

目的探讨荧光原位杂交(FISH)技术在产前诊断中的应用价值。方法采集116名孕妇孕16~23周的羊水标本,应用荧光标记的21号染色体特殊位点探针(21q22,DSCR2)、13号染色体特殊位点探针(13q14,DLEU1)及18号染色体探针、X/Y染色体着丝粒探针(CEP)对未经培养的羊水间期细胞进行FISH检测;同步进行羊水细胞培养,行常规细胞遗传学染色体核型分析。结果 FISH与羊水细胞核型分析相符的染色体数目正常108例,数目异常6例,另各有1例染色体核型分析分别显示为平衡易位、臂内倒位异常,FISH结果显示正常。6例数目异常胎儿引产时抽脐血染色体检查结果与羊水产前诊断结果一致。结论 FISH技术用于快速诊断胎儿常见染色体数目异常,具有简便、快速、特异性强等优点,临床有较高的应用价值。     

应用原位杂交技术比较肺腺癌和肺鳞癌的内在分子机制

目的分析肺腺癌及肺鳞癌肿瘤中染色体异常变化和基因组的改变。探讨肺腺癌及肺鳞癌细胞遗传物质的改变,揭示这两种NSCLC主要亚型发生发展的内在本质及其与临床特征之间的关系。方法应用M-FISH技术,检测肺腺癌及肺鳞癌细胞株各1株,观察其染色体数目及结构畸变情况,应用CGH技术对80例肺腺癌组织80例肺鳞癌组织中所提取的全基因组DNA进行检测,比较这两种类型的NSCLC全基因组的变化。结果M-FISH结果显示肺腺癌及肺鳞癌细胞中存在许多复杂的染色体重排,其中5、6、11、12、17号染色体最频繁参于染色体间的易位。CGH发现,在160例肺癌标本基因组中,最常见的扩增区域足1 q,2 p,3 q,5 p,5 q,7 p,8 q,11 q,12 q,14 q,16 p,17 p.19 p,20 q,21 q,22 q。最常见的缺失区域是2 q.3 p,4 p,5 q,7 q,8 p,9 p.13 q,14 q,17 p。在肺腺癌中最常见的扩增位点足16p13,阳性率达50%.而肺鳞癌中最常见的扩增位点是17q21(45%),并且这两个位点的变异在肺腺癌和肺鳞癌之间有着显著性的差异(P<0.05)。结论M- FISH和CGH技术是研究肺癌基因组变化的强有力的工具,本实验中发现的基因改变町能代表了与肺腺癌及肺鳞癌特有的病理,诊断相关和候选基因。     

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。     

Evidence by spectral karyotyping that 8q11.2 is nonrandomly involved in lipoblastoma

We report two cases of lipoblastoma with chromosome 8-related aberrations, ie, a 92,XXYY,t(7;8)(p22;q11.2)x2 [8]/46,XY[16] in Case 1 and a 46,XY,−8,−13,add(16)(q22),+mar, +r [cp13]/46,XY[7] in Case 2. Using spectral karyotyping and fluorescence in situ hybridization techniques, the karyotype of Case 2 was redesignated as 46,XY, r(8), del(13)(q12), der(16)ins(16;8)(q22;q24q11.2)[cp13]/46,XY[7]. This report delineates a new chromosome rearrangement, ie, der(16)ins(16;8)(q22;q24q11.2) in lipoblastoma, and also confirms the t(7;8)(p22;q11.2), reported only once previously, as a recurrent translocation involved in such a tumor. These findings provide valuable information for clinical molecular cytogenetic diagnosis of lipoblastoma. Furthermore, this report highlights the value of cytogenetic and molecular cytogenetic analysis in differential diagnosis of childhood adipose tissue tumors and adds to the number of lipoblastomas reported with chromosomal abnormalities at 8q11.2.     

荧光原位杂交技术快速诊断胎儿常见染色体数目异常

目的探讨荧光原位杂交（FISH）技术在产前诊断中的应用价值。方法采集116名孕妇孕16~23周的羊水标本,应用荧光标记的21号染色体特殊位点探针（21q22,DSCR2）、13号染色体特殊位点探针（13q14,DLEU1）及18号染色体探针、X/Y染色体着丝粒探针（CEP）对未经培养的羊水间期细胞进行FISH检测;同步进行羊水细胞培养,行常规细胞遗传学染色体核型分析。结果 FISH与羊水细胞核型分析相符的染色体数目正常108例,数目异常6例,另各有1例染色体核型分析分别显示为平衡易位、臂内倒位异常,FISH结果显示正常。6例数目异常胎儿引产时抽脐血染色体检查结果与羊水产前诊断结果一致。结论 FISH技术用于快速诊断胎儿常见染色体数目异常,具有简便、快速、特异性强等优点,临床有较高的应用价值。     

Simultaneous translocation of both TCR Loci (14q11) with rare partner loci (Xq22 and 12p13) in a case of T-lymphoblastic leukemia

The most common recurrent cytogenetic abnormalities in T-lymphoblastic leukemia (T-acute lymphoblastic leukemia [T-ALL]) involve T-cell receptor (TCR) loci and a variety of partner genes, including HOX11, HOX11L2, MYC, and TAL1. In this report, we present a rare case involving simultaneous translocation of the TCR α/δ loci with different partner loci (Xq22 and 12p13); this resulted in a poor prognosis. Chromosomal analysis showed 46,Y,t(X;14)(q22;q11.2),t(12;14)(p13;q11.2) and FISH analysis by using a T-cell receptor alpha delta DNA probe, Split Signal (DakoCytomation, Denmark), showed translocations at the same TCR α/δ locus on both chromosomes. FISH with 2 bacterial artificial chromosome clones showed break apart signal, which suggests involvement of the IRS4 gene. To our knowledge, this is the first report of T-ALL in which both TCR α/δ loci were translocated with different partner loci, and 1 of the partner loci, Xq22, was a rare translocation partner locus that included IRS4 gene.     

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment.     

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.     

Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification

BACKGROUND: Approximately 99% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11.2-q13, uniparental disomy for chromosome 15 (UPD15), or imprinting center defects affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of genomic abnormalities depends on the parent of origin. Both disorders are characterized at the molecular level by abnormal methylation of imprinted regions at 15q11.2-q13. Other rare chromosome 15 rearrangements and a few smaller atypical deletions associated with abnormal methylation patterns also have symptoms overlapping with either PWS or AS. METHODS: We designed a methylation-specific melting analysis (MS-MA) method for a rapid screening of PWS/AS and evaluated methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for diagnosis of PWS/AS associated with deletions, UPD15, or rare duplications. Forty-nine previously genotyped samples were tested by MS-MA. We also tested 26 MS-MA genotyped samples and 1 additional sample with rare duplication of chromosome region 15q11-q12. RESULTS: PWS/AS genotyping results obtained by MS-MA and by MS-MLPA were fully concordant. In addition, MS-MLPA was superior in detecting deletions/rare duplications, possible UPD15, or imprinting center defects, which were usually determined by a laborious fluorescence in situ hybridization method or by chromosomal segregation analysis for the parental-origin using short-tandem repeat makers. CONCLUSIONS: MS-MA appears to be an efficient primary method to diagnose PWS/AS, and use of the quantitative MS-MLPA method provides detailed information about deletions, rare duplications, and possibly UPD.     

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.     

常规细胞遗传学检查联合多重荧光原位杂交技术确定急性白血病患者复杂核型异常

目的 探讨多重荧光原位杂交(M-FISH)技术在急性白血病(AL)患者复杂核型异常和标记染色体检测中的应用价值.方法 对11例常规R显带检测显示具有复杂核型异常和标记染色体的AL患者应用M-FISH技术确定复杂染色体重排和标记染色体的组成,识别并确定微小易位和隐匿易位.结果 11例AL患者应用常规细胞遗传学(CC)技术共检出27种染色体数目异常和41种结构异常.CC技术检出的3种染色体增加和9种染色体丢失以及12种结构异常与M-FISH分析结果一致,CC技术检出的15种染色体丢失经M-FISH证实为衍生染色体,M-FISH还检出3种CC检查未发现的染色体数目增加.CC检查所检出的其余29种结构异常(包括17种标记染色体)的性质被M-FISH进一步明确.M-FISH共检出33种结构重排,有6种异常未见文献报道,分别为t(5q-;16)(?q14;q24)、der(9)(Y::9::Y::9)、der(7)(7::8::9)、ins(20;21)、der(11)(11::21::20)和der(3)t(3p-;13)(3p-;q21),这些复杂染色体重排主要由染色体不平衡易位所致.复杂核型异常几乎涉及所有染色体,在AML患者以涉及17、5、7、15和11号染色体的异常较为常见;在ALL患者则以涉及8、9、14和22号染色体的异常较为多见.结论 联合应用CC和M-FISH检查可以提高CC核型分析的分辨率,对伴复杂核型和标记染色体的AL具有一定的临床应用价值.     

The role of fluorescence in situ hybridization technologies in molecular diagnostics and disease management.

Large genomic changes, such as aneuploidy, deletions, and other chromosomal rearrangements, have long been associated with pregnancy loss, congenital abnormalities, and malignancy. These genomic changes are quantitative, unambiguous, and fundamental in the transition of normal cells to abnormal ones. Detection of these large genetic changes has an increasingly important role in determining patient diagnosis and care, including therapeutic selection. We have developed two major product platforms that assess genomic changes at various levels of resolution. Fluorescence in situ hybridization (FISH) techniques and the related technology of array-based comparative genomic hybridization (CGH) allow detection of genesized or larger alterations in the genome. FISH is a robust DNA probe technology that can measure both balanced and unbalanced genomic changes on a cell-by-cell basis. In most instances, it is not dependent on metaphase chromosomes, and it is widely used in clinical diagnostics. Array-based CGH has much greater multiplexing capabilities than FISH. This technology has the potential to examine many regions of the genome simultaneously for changes in DNA copy number and identify complex patterns of gains and losses within the genome. In this article, we review several of the current medical applications of FISH and discuss such advanced techniques as CGH and array-based CGH.     

荧光原位杂交技术检测骨髓增生异常综合征患者5、7、8号染色体异常及临床意义

目的用荧光原位杂交（FISH）技术分析骨髓增生异常综合征（MDS）患者的染色体改变及预后。方法用常规细胞遗传学分析和FISH法分析37例MDS患者8、5、7号染色体的异常变化。用SPSS11．5统计软件，对患者的遗传学异常与疾病转归、预后之间关系进行相关性检验。结果检出染色体异常21例（56．8％），其中复杂异常6例（16．2％），8号染色体异常9例（24．3％），-5／5q-异常2例（5．4％），-7／7q-异常2例（5．4％）。平均随访12个月，1例失访，22例存活，14例死亡，12例转变为急性白血病。复杂核型与MDS的急性白血病转化及死亡密切相关；8号染色体三体和-7／7q-与死亡相关。结论FISH能敏感地检测出小克隆的异常，应用多种探针并结合染色体检测能较准确判断MDS患者的预后，异常核型比例高提示预后差。     

CD5-negative blastoid variant mantle cell lymphoma with complex CCND1/IGH and MYC aberrations

The coexistence of CCND1/IGH and MYC rearrangements in mantle cell lymphoma (MCL) is a rare finding associated with a very poor prognosis. In this study, a patient with blastoid variant (MCL) is reported. The disease was clinically aggressive and refractory to chemotherapy, and the patient only survived for 1 month following diagnosis. Conventional cytogenetic study, FISH, and multicolor FISH (mFISH) demonstrated the involvement of the BCL1/CCND1 locus in a complex translocation, t(3;11)(q25;p15)t(11;14)(q13;q32). In addition, subclonal abnormalities in the 8q24 region, manifested as a t(8;14)(q24;q32)/MYC rearrangement, were identified. To the best of our knowledge, this is the first MCL case in Korea bearing these complex genomic aberrations.     

原发性血小板增多症的细胞遗传学特征

目的研究原发性血小板增多症(ET)患者的细胞遗传学特征。方法应用经典细胞遗传学(CC)方法R显带技术研究98例初诊ET患者的染色体异常,进一步应用SpectrumRed标记的8号染色体着丝粒DNA探针和间期荧光原位杂交技术(FISH)检测50例患者+8的发生率。结果CC分析98例ET患者除2例无分裂相外,仅6例(6.12%)发现染色体异常:1例13q-、2例5q-、1例7q-、1例der(14)t(1;14)和1例Rob(13;14)。FISH分析50例患者中1例+8。结论CC技术检测ET患者染色体异常发生率为6.25%,且缺乏特征性染色体异常。+8发生率低。