一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

Role of multicolor fluorescence in situ hybridization (FISH) in simultaneous detection of probe sets for chromosome 18, X and Y in uncultured amniotic fluid cells.

Major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. The purpose of this study was to evaluate the role of multicolor fluorescence in situ hybridization in simultaneous detection of probe sets for chromosome 18, X, and Y in uncultured amniotic fluid cells as a safer alternative method for aneuploidy detection prenatally. Fifty amniotic fluid samples were analyzed by FISH and standard cytogenetics. Mean time to obtain results was three days for fluorescence in situ hybridization and 20 days for karyotype. Fluorescence in situ hybridization was informative in 43 samples (86%), and within this group, two aneuploidies were correctly identified. This evaluation demonstrates that FISH with X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory.     

Detection of monosomy 7 by fluorescenceIn situ hybridization in acute nonlymphocytic leukemia and myelodysplastic syndrome

Summary Fluorescencein situ hybridization (FISH) with a chromosome 7 specific alpha satellite DNA probe was used to detect monosomy 7 in interphase and metaphase cells obtained from patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). Chromosome analysis revealed monosomy 7, either alone or as part of a complex chromosome abnormality, in all cell samples. FISH analyses of 12 marrow samples and a blood sample using a chromosome 7 specific alpha satellite DNA probe revealed a single fluorescence spot in 80.5–97.5% of interphase cells indicating monosomy 7. In contrast, 83.5–92.0% of the same cells had two copies of chromosome 17 as two fluorescent spots were detected using a chromosome 17 specific alpha satellite DNA probe used as a positive control. The proportion of interphase cells with monosomy 7 did not correlated with the percentage of metaphase cells with monosomy 7 detected by conventional karyotyping or with the percentage of blast cells in the bone marrow.     

Amniotic fluid interphase fluorescence in situ hybridization (FISH) for detection of aneuploidy; experiences in 130 prenatal cases

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, 21, and sex chromosomes X and Y. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. We retrospectively reviewed 130 amniotic fluid interphase FISH analyses from January 1997 to December 2001. The review was done in order to assess the role of interphase FISH among the patients who were at the risk of fetal aneuploidies. The sample was considered to be aneuploid when 70% of or more than the total number of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. All of 130 cases but one met the criteria. The results were considered as informative and they were obtained in 24-48 hr. The overall detection rate for aneuploidies was 100% (2 cases of trisomy 21, 2 cases of trisomy 18, and 1 case of Turner syndrome). In comparison to cytogenetics, the rates of both sensitivity and specificity were 100%. The experiment demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. The experiment can also serve as an adjunctive test to help cytogenetics to reduce significant amount of emotional stress of patients and physicians through early decision making process.     

Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase cells

Benzene is an established human leukemogen that increases the level of chromosome aberrations in lymphocytes of exposed workers. Numerical aberrations (aneusomy) can be observed by fluorescence in situ hybridization (FISH) in both interphase and metaphase cells. Whereas interphase FISH allows nondividing cells to be analyzed, one advantage of metaphase FISH is that it can also detect structural changes. The present study compares the abilities of metaphase and interphase FISH to detect aneusomy of chromosomes 7 and 8 in healthy benzene-exposed human subjects. Metaphase and interphase cells from the peripheral blood of 43 workers exposed to benzene (median = 31 ppm, 8-hr TWA) and 44 frequency-matched controls were analyzed by FISH. Normal diploid cells contained two hybridization signals, whereas those that were potentially monosomic contained one, trisomic 3 and tetrasomic 4. The frequency of cells with one hybridization signal for chromosome 7 in metaphase spreads rose from 2.72 +/- 0.19 (%, mean +/- SE) in controls to 3.79 +/- 0.63 in workers exposed to 31 or fewer ppm benzene and 5.9 +/- 0.85 in those exposed to more than 31 ppm (P(trend) < 0.0001). No similar dose-dependent increase in the frequency of cells with one hybridization signal was observed by interphase FISH, probably because of probe overlap artifact. Although significant dose-dependent increases in the frequency of cells with three hybridization signals for chromosome 7 were detected by both methods in the higher-exposed group, a larger, more significant difference was detected by metaphase FISH between controls and workers exposed to 31 or fewer ppm. Similar data were obtained for chromosome 8. Interphase and metaphase FISH were moderately correlated for three hybridization signals but not for one hybridization signal in chromosome 7 or 8. In general, metaphase FISH was more sensitive in detecting both monosomy and trisomy in the lymphocytes of exposed workers.     

Fluorescence in situ hybridization (fish): A user's guide to optimal preparation of cytologic specimens

Fluorescence in situ hybridization (FISH) is a reliable method for tagging centromeric regions of specific chromosomes in interphase nuclei. Not only is FISH useful for chromosome enumeration, but as region-specific chromosome probes are developed, the clinical applications and potentials for use by pathologists are extensive. This technique lends itself particularly to use in cytology preparations because the cells are disaggregated and monolayer preparations yield excellent technical hybridization results. Over a 7-mo period we processed cytologic samples in an attempt to define and outline a method for optimal specimen processing for FISH use in cell suspensions, techniques applicable to all fresh cytology specimens which can also be used for the processing of surgical pathology aspirates and other material. All samples should be promptly processed to ensure specimen viability, and triaged on an individual basis to ensure preparation of moderately cellular monolayered cytospins. Equivalent nuclear probe signals have been obtained with several sample fixation methods air-drying, 95% ethanol, methanol (Diff-Quik fixative), and Carnoy's solution. No difference was noted in the nuclear probe signals or specimen adhesion on positively charged or noncharged slides. After initial fixation our slides remained at room temperature until FISH was performed, without any adverse effects. A short digestion with proteinase K and subsequent rehybridization yielded positive results on samples that originally yielded poor nuclear probe signals. © 1995 Wiley-Liss, Inc.     

Analysis of chromosome 12 aneuploidy in interphase cells from human male germ cell tumors by fluorescence in situ hybridization.

The i(12p) marker chromosome has been found to be a highly nonrandom chromosome abnormality associated with germ cell tumors (GCTs). We have previously shown that a chromosome 12 centromere specific alpha-satellite DNA probe detects the i(12p) by virtue of differences in the size of the signal originating from the i(12p) and normal chromosome 12 centromeres after fluorescence in situ hybridization (FISH) in metaphase and interphase cells of cultured GCT cell lines. We have now extended this analysis to 72 fresh GCT tumor biopsy specimens. Banded cytogenetic analysis was attempted on each of these tumors, 45 of which were found to be clonally abnormal. Data on i(12p) and chromosome 12 copy number obtained by FISH agreed well with those obtained by cytogenetic analysis. In addition, the FISH method made possible the detection and determination of i(12p) and the chromosome 12 copy number in cases in which conventional cytogenetic analysis was unsuccessful. We found the incidence of i(12p) in seminomas to be low (7%) compared to that in nonseminomas (75%) when tumor biopsy specimens were studied by FISH. Our results show that the FISH technique can be used reliably for detection of the diagnostically and prognostically useful i(12p) marker in GCT tumor biopsy specimens.     

Detection of karyotype changes in interphase cells: oligonucleotide-primed in situ labelling versus fluorescence in situ hybridization

Interphase cytogenetics is a rapidly developing technique which is usually performed by fluorescence in situ hybridization (FISH). Recently, oligonucleotide-primed in situ synthesis (PRINS) has become established as a method of labelling centromeric regions of chromosomes in metaphase spreads. We tested the suitability of PRINS in detecting the exact copy number of chromosomes 1, 3, 7 and 8 in intact interphase cells of 17 cytological preparations derived from normal and neoplastic tissues. Control procedures consisted in preparation of metaphase spreads of lymphocytes of healthy donors, conventional cytogenetics in some of the specimens, and omission of the primers or Taq polymerase from the reaction mixture. All specimens were additionally examined by FISH and analysed blind by two experienced observers. Both PRINS and FISH revealed a corresponding distribution of hybridization signals for all chromosomes examined in specimens of normal bone marrow (n = 5), normal liver cells (n = 5), three samples of acute nonlymphocytic leukaemia in which conventional chromosome analyses had shown monosomy 7 or trisomy 8, and in four hepatocellular carcinomas that displayed trisomy 1. Overall, statistical analysis revealed no significant difference in the signal distribution between the two techniques. Our results demonstrate that PRINS is as reliable as FISH for detecting chromosome copy numbers in interphase nuclei of intact cells. The PRINS method, however, is easier to perform, faster and less expensive, holding great potential for future applications in diagnostic pathology.     

The Art and Applications of Fluorescence In Situ Hybridization in Endocrine Pathology

Fluorescence in situ hybridization (FISH) or molecular cytogenetics is currently recognized as a reliable, sensitive, and reproducible technique for identifying the copy number and structure of chromosomes. FISH combines molecular genetics with classic cytogenetics and allows simultaneous morphologic evaluation on a single slide. Centromeric DNA probes are used to detect specific chromosomes and telomeric probes to demonstrate all chromosomes. Sequence-specific probes can localize in situ a single gene copy on a specific chromosome locus. FISH allows cytogenetic investigation of metaphase spreads and interphase nuclei. Several protocols have been proposed to analyze preparations from fresh samples or archival material. Comparative genomic hybridization (CGH) is a novel cytogenetic technique, which combines FISH with automatic digital image analysis. Comparative analysis of the hybridization products of tumor DNA and reference DNA with normal metaphase chromosomes, each labeled with color different fluorochrome, can retrieve chromosomal imbalances of the entire genome in a single experiment. FISH and CGH are powerful morphologic tools in understanding physiologic mechanisms and in resolving problems of the pathogenesis of several diseases. These techniques shed light on the cytogenetic background in many endocrinological disorders, providing a better understanding of the activities and alterations of endocrine cell function.     

Increased amplification proximal to the MLL gene in acute myeloid leukemia

Amplification of the chromosomal region 11q23 encompassing the MLL gene has been observed in patients with acute myeloid leukemia (AML). Patients with this abnormality often have a poor response to chemotherapy and short survival. We have studied the regions flanking the MLL gene using 8 bacterial artificial chromosome (BAC) probes and dual color fluorescence in situ hybridization (FISH) analysis. Two contigs of BAC probes flanking the MLL gene, were constructed by standard primer walking and BAC end sequencing. For comparison, metaphase chromosome preparations were also hybridized with a commercial MLL specific probe. Metaphase chromosomes were prepared from the bone marrow sample of an 80-year old female patient with AML-M1 and the cytogenetic aberration der(11)hsr(11) (q23). FISH analysis on metaphase chromosomes showed amplification on the homogeneously staining region (hsr) and marker chromosomes for both the MLL gene and the BAC probes. Using dual color FISH, probes proximal to MLL showed greater amplification than those distal to MLL, as represented by additional red signals on both metaphase and interphase chromosomes. Ratios of the copy numbers of the BAC probes relative to the chromosome 11 centromere copy number confirmed a higher copy number for probes proximal to MLL. These results suggest that other gene(s) proximal to MLL could be the target gene(s) of amplification in this case and not the MLL gene as previously assumed.     

Case with intrauterine fetus death: interphase fluorescence in situ hybridization using buccal cells is useful for examining chromosomal abnormalities when placental villus

The proband was a male fetus who died at 18 weeks of gestation. The fetus had growth retardation, hydrocephalus, exophthalmos, and micrognathia. The placental villus was not available. We performed interphase fluorescence in situ hybridization (FISH) using buccal cells of the fetus. The FISH using centromere specific probes for chromosome 7, 8 and 18, and RB1 gene (13q14)-specific probe showed three signals for each chromosome. The sex chromosome composition was XXY by FISH using centromere-specific probes for X and Y chromosomes. Thus, the fetus was diagnosed with triploidy syndrome. This report suggested that interphase FISH using buccal cells is useful for examining chromosomal abnormalities in intrauterine fetal death when placental villus is not available.     

Characterization of the topoisomerase I locus in human colorectal cancer

DNA topoisomerase I (topo I) is the principle target for Camptothecin and its analogues. The topo I gene is located on chromosome 20q11.2-q13.1 and variation in topo I gene copy number has been shown to have impact on the in vitro sensitivity to topoisomerase I inhibitor chemotherapy. Fluorescence in situ hybridization (FISH) was used to detect and compare the TOPO I gene copy number between metaphase and interphase nuclei in a panel of 7 colorectal cancer cell lines. TOPO I gene copy number varied from 2 to 8 between cell lines, and signal in interphase nuclei demonstrated a linear relationship with that detected in metaphase nuclei. The structure of gene amplification included isochromosome formation, amplicon extension, and marker chromosome generation. Comparative genomic hybridization (CGH) was then used to further define the region of gain on chromosome 20. The region of gain contained the topo I gene and involved nearly all of 20q in most cases. This demonstrates a high degree of intrinsic variation in topo I gene copy number and the involvement of a 20q amplicon in colorectal cancer, which may have important implications for colorectal tumorigenesis and the use of chemotherapy.     

间期FISH分析中细胞类型及探针的选择

荧光原位杂交已成为医学遗传学领域中一项极为重要的研究手段。为了摸索不同来源的产前诊断材料的间期核进行荧光原位杂交的可行性，选择了α重复序列ＤＮＡ及ＣｏｓｍｉｄＤＮＡ为探针，分别与未培养的绒毛间质细胞、羊水细胞及外周血白细胞的间期核杂交。结果表明：各种材料的间期核均见杂交信号，１３／２１α重复序列探针用于未培养的绒毛间质细胞及外周血白细胞的杂交信号最为特异，能有效的检出染色体非整倍体，羊水细胞则远不如绒毛细胞，绒毛组织是最理想的产前诊断材料。     

人21号全染色体涂染探针的制备及其唐氏综合征诊断的应用研究

目的人21号染色体DNA涂染探针的制备及其应用于唐氏综合征诊断的研究。方法将显微分离的人21号染色体DNA进行简并寡核苷酸引物PCR（Degenerate Oligonucleotide Primed—PCR,DOP—PCR）扩增并标记制备成杂交探针后,与15例唐氏征疑似患者的外周血细胞核染色体进行荧光原位杂交（Fluorescence in situ hybridization,FISH）分析;同时以常规核型分析进行确诊并评估FISH结果。结果FISH分析诊断结果与常规核型分析一致,其中8例为非唐氏征患者,7例为唐氏征患者,准确率为100％,且非唐氏征患者与唐氏征患者细胞核中21号染色体的检出率分别高达99．12％和99．08％。结论制备的人21号全染色体DNA涂染探针能精确检测人类中期和间期细胞核中21号染色体的数目,该探针可用于唐氏综合征的诊断。     

Fluorescence in situ hybridization to determine engraftment status after murine bone marrow transplant.

The mouse Y-specific DNA sequence pY2 was used as a probe for fluorescence in situ hybridization (FISH) to evaluate murine hematopoietic tissues after sex-mismatched bone marrow transplant (BMT). The pY2 probe was localized to the long arm of the Y chromosome on BM metaphases. Hybridization of pY2 in FISH of interphase cells from BM, spleen, and thymus after BMT was compared with Southern blot analysis; both methods gave comparable results. Only FISH was able to analyze post-BMT peripheral blood (PB) samples successfully, and provides a useful method for following engraftment status in the mouse on an ongoing basis.     

实体瘤间期细胞制备技术对荧光原位杂交效果的影响

目的将改进的实体瘤间期细胞制备方法应用于荧光原位杂交,为实体瘤间期细胞的制备建立技术平台。方法采用随机引物法标记3号染色体着丝粒探针,分析实体瘤不同间期细胞制备技术对荧光原位杂交效果的影响。结果6种制片方法各具特点,根据其在应用方面有不同侧重,可采用不同制片方法。胶原酶法制片的荧光原位杂交效果最佳。对于冰冻组织或较小的组织,可采用印片法制片。结论适当的制片方法,结合荧光原位杂交,为实体瘤的染色体研究和诊断应用奠定了技术基础。     

Detection of trisomy 8 in philadelphia chromosome-positive CML patients using conventional cytogenetic and interphase fluorescence in situ hybridization techniques and its relation to c-myc involvement

Trisomy 8 (+8) is a common clonal evolution marker for progression in chronic myelogenous leukemia. The relationship of +8 to various stages of t(9;22) leukemias is not firmly established. To explore this association we examined bone marrow (BM) cells from 10 Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) patients in different stages of the disease, using conventional cytogenetic technique(CCT) and interphase fluorescence in situ hybridization (FISH). FISH detection of chromosome 8 was accomplished using the D8Z2 (Oncor) probe specific for the centrometric region of chromosome 8. Five hundred interphase nuclei were counted for each patient. Three of the 10 patients were selected for detection of c-myc gene locus located in the 8q24.2-24.3 region using the L     

恶性血液病8号染色体数目异常的间期荧光原位杂交检测

目的　探讨间期荧光原位杂交 (fluorescenceinsituhybridization ,FISH)技术在检测恶性血液病 8号染色体数目异常中的价值。方法　采用常规细胞遗传学 (conventionalcytogenetics ,CC)和 8号染色体着丝粒特异性探针间期FISH技术对 8例CC检测显示 8号染色体数目异常的急性髓细胞样白血病患者、10例慢性髓细胞样白血病加速期或急变期患者和 3名正常人骨髓进行 8号染色体数目检测。结果9例CC检测为三体 8的患者中 ,FISH检测结果均与其一致 ,其中例 5经CC检测仅发现存在二体 8、三体8和四体 8克隆 ,而FISH检测不但证实了三体 8和四体 8克隆的存在 ,还发现存在一个较小的五体 8克隆。例 3和例 17经CC检测只发现一个细胞有三体 8,无法确定是否为三体 8克隆性畸变 ,FISH检测证实有三体 8克隆存在。例 9经CC检测未发现三体 8,FISH检测发现有三体 8克隆存在。与CC检测结果相比 ,除例 16三体 8检出率FISH结果明显高于CC检测结果外 ,其余均低于或接近CC检测结果。结论　间期FISH技术对检测 8号染色体数目异常具有重要价值 ,当CC检测正常、不肯定或中期分裂相质量差、数量少时作用更大 ,是CC的重要补充。     

FISH analysis in chromophobe renal-cell carcinoma

Loss of chromosomes 1, 2, 6, 10, 13, 17, and 21 is a characteristic finding in chromophobe renal-cell carcinoma (ChRCC). Previously, cytogenetic and molecular genetic techniques were used in demonstrating the chromosomal monosomies in ChRCCs. We performed interphase fluorescent in situ hybridization (FISH) using centromeric probes for chromosomes 1, 2, 6, and 10 on touch imprint smears from six histologically proven ChRCCs. All six ChRCC tumors showed one FISH signal corresponding to one copy number for each of these chromosomes. The percent cells with one FISH signal ranged from 48-88% (chromosome 1), 36-89% (chromosome 2), 26-98% (chromosome 6), and 64-99% (chromosome 10). In addition, 3 of the 6 cases were further studied with centromeric probes for chromosomes 13, 17, and 21. All three revealed monosomy of these three chromosomes. We conclude that interphase FISH performed on touch imprint smears is a relatively simple, rapid, and reliable method for detecting chromosome abnormalities which are specific for ChRCCs.     

DiGeorge/velocardiofacial syndrome: FISH studies of chromosomes 22q11 and 10p14, and clinical reports on the proximal 22q11 deletion

Interphase fluorescence in situ hybridization (FISH) analysis can provide rapid preliminary analysis of chromosome aneuploidy from direct amniocyte and chorionic villus sample (CVS) preparations. Typically, interphase FISH is used in screening for numerical abnormalities of chromosomes X, Y, 13, 18, and 21. More recently, FISH probe sets became available for the subtelomeric region of each chromosome, allowing screening for terminal chromosome rearrangements. The purpose of the current study was to evaluate the use of dual-color interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in 14 pregnancies from 12 different translocation carriers. Interphase FISH analysis was performed on direct CVS or amniocyte preparations from 12 reciprocal translocation and two Robertsonian translocation pregnancies with the appropriate chromosome-specific subtelomere probes for each chromosome involved in the translocation. Analysis of the interphase FISH probe signals predicted balanced or normal segregants in each case, thus rapidly excluding a chromosomally unbalanced segregant. Subsequent metaphase analysis showed normal karyotypes in seven fetuses and balanced translocations in the remaining seven. This series illustrates the utility of interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in cases of parental reciprocal translocations and Robertsonian translocations.