Novel karyotypic changes detected by comparative genomic hybridization in a case of congenital cervical immature teratoma

BACKGROUND: Cervical immature teratoma is a rare congenital tumor, and very few cases have been studied cytogenetically. CASE: In this article, we describe a case of this tumor type and present the findings of the karyotype of the lesion, which was performed with the bacterial artificial chromosome arrays using the comparative genomic hybridization method. The chromosomal abnormalities that we found included an amplification on 1p21.1, a 9p22 deletion, and a 1-copy gain of 17q21.33. CONCLUSIONS: None of the identified chromosomal aberrations have been previously associated with congenital extragonadal teratomas. Important genes that lie in these DNA regions may be implicated in the pathogenesis of congenital teratomas.     

Genomic characterization of some Iranian children with idiopathic mental retardation using array comparative genomic hybridization

BACKGROUND:

Mental retardation (MR) has a prevalence of 1-3% and genetic causes are present in more than 50% of patients. Chromosomal abnormalities are one of the most common genetic causes of MR and are responsible for 4-28% of mental retardation. However, the smallest loss or gain of material visible by standard cytogenetic is about 4 Mb and for smaller abnormalities, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH) should be used. It has been shown that 15-25% of idiopathic MR (IMR) has submicroscopic rearrangements detectable by array CGH. In this project, the genomic abnormalities were investigated in 32 MR patients using this technique.

MATERIALS AND METHODS:

Patients with IMR with dysmorphism were investigated in this study. Karyotype analysis, fragile X and metabolic tests were first carried out on the patients. The copy number variation was then assessed in a total of 32 patients with normal results for the mentioned tests using whole genome oligo array CGH. Multiple ligation probe amplification was carried out as a confirmation test.

RESULTS:

In total, 19% of the patients showed genomic abnormalities. This is reduced to 12.5% once the two patients with abnormal karyotypes (upon re-evaluation) are removed.

CONCLUSION:

The array CGH technique increased the detection rate of genomic imbalances in our patients by 12.5%. It is an accurate and reliable method for the determination of genomic imbalances in patients with IMR and dysmorphism.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.     

Arcyptera fusca and Arcyptera tornosi repetitive DNA families: whole-comparative genomic hybridization (W-CGH) as a novel approach to the study of satellite DNA libraries

Whole-comparative genomic hybridization (W-CGH) has been used to exemplify a simple methodology which allows identifying and mapping whole genome differences for highly repetitive DNA sequences between two related species of unknown genomic background. The use of this technique to the species binomy Arcyptera fusca/Arcyptera tornosi has allowed the identification of different DNA families mainly concentrated within the para-/peri-centromeric and distal heterochromatic regions of different chromosomes, which are differentially expanded in both genomes. Additionally, W-CGH allowed chromosome mapping of particular euchromatic regions immersed in the chromosome arms which have been affected by processes of DNA amplification and losses. A molecular approach was also conducted to analyse satellite DNA families in these species. We have found three different families showing an unequal representation in both species. Two of these families showed a centromeric location (EcoRV-390CEN and Sau3A-419CEN), whereas the last one was located at distal heterochromatic regions (Sau3A-197TEL). As A. fusca is a widely distributed species represented in most European high mountains, whereas A. tornosi is an endemic species represented in the Iberian Peninsula, the differences and resemblances reported here offer a good basis to support a close evolutionary relationship between both of the actually isolated species. Finally, W-CGH allowed identification of an asynchronic pattern of heterochromatin condensation through early prophase (characteristic in both species) which is uncommon or probably has been poorly analysed within classical early condensing chromosome domains through meiosis. The congruence of the obtained cytological and molecular results is analysed in light of the ancestral genome relationship between both species.     

Chromosome 11 aneuploidy detected by fluorescent in situ hybridization (FISH) in breast cancer: relation with progesterone receptor expression

Progesterone receptor (PR) expression is known to be impaired in breast cancer. As the PR gene is located on chromosome 11 which is also often affected, we studied their relationship in 15 patients with breast carcinoma. Tumoural imprints were used for PR immunocytochemistry and for FISH with chromosome 11 centromeric probes. Distribution profiles of chromosome 11 number in PR+ and PR- cell populations were examined. No difference in the number of chromosome 11 was found between PR+ and PR- breast tumours. Thus, loss of PR expression in breast cancer cannot be explained only by loss of chromosome 11; other genetic or non-genetic mechanisms should be advanced.     

Differential gene alteration among hepatoma cell lines demonstrated by cDNA microarray-based comparative genomic hybridization

We assayed chromosomal abnormalities in hepatoma cell lines using the microarray-based comparative genomic hybridization (array-CGH) method and investigated the relationship between genomic copy number alterations and expression profiles in these hepatoma cell lines. We modified a cDNA array-CGH assay to compare genomic DNAs from seven hepatoma cell lines, as well as DNA from two non-hepatoma cell lines and from normal cells. The mRNA expression of each sample was assayed in parallel by cDNA microarray. We identified small amplified or deleted chromosomal regions, as well as alterations in DNA copy number not previously described. We predominantly found alterations of apoptosis-related genes in Hep3B and HepG2, cell adhesion and receptor molecules in HLE, and cytokine-related genes in PLC/PRF/5. About 40% of the genes showing amplification or loss showed altered levels of mRNA (p < 0.05). Hierarchical clustering analysis showed that the expression of these genes allows differentiation between alpha-fetoprotein (AFP)-producing and AFP-negative cell lines. cDNA array-CGH is a sensitive method that can be used to detect alterations in genomic copy number in tumor cells. Differences in DNA copy alterations between AFP-producing and AFP-negative cells may lead to differential gene expression and may be related to the phenotype of these cells.     

Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5′-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.     

Novel clusters of highly expressed genes accompany genomic amplification in breast cancers

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.     

Establishment of a multi-color genomic in situ hybridization technique to simultaneously discriminate the three interspecific hybrid genomes in gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.     

Characterization of serogroup C meningococci isolated from 14 provinces of China during 1966-2005 using comparative genomic hybridization

Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. In China, serogroup A strains were responsible for over 95% of the cases, while serogroup B strains were mainly the cause of localized outbreaks and sporadic cases. Before 2003, serogroup C strains were only recovered from a few sporadic cases. However, a sudden increase in the number of cases due to serogroup C strains occurred during 2003-2005 in Anhui Province, China. Many cases were found in other provinces at the same time. Multilocus sequence typing (MLST) results indicated that the unique sequence type 4821 clone meningococci, a new hyper-virulent lineage, was responsible for the serogroup C meningitis outbreaks. We have completed the project of sequencing the whole genome of the Chinese N. meningitidis serogroup C representative isolate 053442. We fabricated a whole-genome microarray of N. meningitidis isolate 053442 and analyzed the genome composition differences among 81 serogroup C isolates which were isolated from 14 provinces of China during 1966-2005. The comparative genomic hybridization (CGH) result shows that the genome compositions of nearly all serogroup C isolates are similar to that of 053442. The products of many absent open reading frames (ORFs) are conserved hypothetical proteins. The results will provide a valuable resource from which one can analyze the genome composition and genetic background of serogroup C meningococci in China.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.     

Chromosome imbalances in oligodendroglial tumors detected by comparative genomic hybridization

Seven well-differentiated oligodendrogliomas, 16 anaplastic oligodendrogliomas and two cases of oligoastrocytomas were investigated by comparative genomic hybridization (CGH) on frozen tissue samples. The most frequent losses found involved 1p and 19q in 32% of cases. Loss of 9p was observed during malignant progression in 25% of anaplastic oligodendrogliomas. In two anaplastic oligodendrogliomas gain of 1q was found. The frequent losses of chromosome 16 and 22 have not been reported previously. These results underscore that CGH is a powerful tool for the classification of gliomas complementing the traditional histopathological approach.     

Chromosomal mapping of T-DNA inserts in transgenicPetunia byin situ hybridization

The chromosomal location of T-DNa inserts in ten independently derived and confirmed transgenic plants ofP. hybrida was detected byin situ hybridization. Nine transgenic plants had the T-DNA inerts at single sites distributed among each of the seven chromosomes; in one plant the T-DNA inserts were detected on two different chromosomes. Although the T-DNA inserts were integrated randomly among the chromosomes, seven of the 11 total inserts were located at or near the telomere. Thus, T-DNA inserts appear to have potential for tagging chromosomes and chromosome fragments.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

A genome-wide survey of copy number variation regions in various chicken breeds by array comparative genomic hybridization method

The discovery of copy number variation (CNV) in the genome has provided new insight into genomic polymorphism. Studies with chickens have identified a number of large CNV segments using a 385k comparative genomic hybridization (CGH) chip (mean length >140 kb). We present a detailed CNV map for local Chinese chicken breeds and commercial chicken lines using an Agilent 400k array CGH platform with custom-designed probes. We identified a total of 130 copy number variation regions (CNVRs; mean length = 25.70 kb). Of these, 104 (80.0%) were novel segments reported for the first time in chickens. Among the 104 novel CNVRs, 56 (53.8%) of the segments were non-coding sequences, 65 (62.5%) showed the gain of DNA and 40 (38.5%) showed the loss of DNA (one locus showed both loss and gain). Overlapping with the formal selective sweep data and the quantitative trait loci data, we identified four loci that might be considered to be high-confidence selective segments that arose during the domestication of chickens. Compared with the CNVRs reported previously, genes for the positive regulation of phospholipase A2 activity were discovered to be significantly over-represented in the novel CNVRs reported here by gene ontology analysis. Availability of our results should facilitate further research in the study of the genetic variability in chicken breeds.     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

The allopolyploid origin of Narcissus obsoletus (Alliaceae): identification of parental genomes by karyotype characterization and genomic in situ hybridization

Karyological and genomic in situ hybridization (GISH) approaches provided evidence of the parentage and origin of the hybrid species Narcissus obsoletus. Here, we demonstrate that the putative parental species, N. serotinus L. and N. elegans (Haw.) Spach, recently proposed because of their intermediate morphological traits, have participated in the hybridization process forming this taxon. Karyotype characterization of parental genomes in populations from S Spain and N Morocco has revealed differences in chromosome length and karyotype asymmetry, highlighting their diploid nature. Multicolour GISH on metaphase plates of N. obsoletus, with N. serotinus and N. elegans DNA used as probes, showed differential fluorescent staining of 10 and 20 chromosomes from parental genomes, respectively. Both parental genomes were detected in the allopolyploid, albeit in a duplicated manner. Secondary hybridization between N. obsoletus and N. serotinus was also detected karyologically. Little karyological differentiation between different geographic regions was found in either N. serotinus or N. obsoletus. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 477–498.     

拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

利用生物信息数据库筛选乳腺癌CGH微阵列BAC克隆

利用CCAP数据库和UCSC数据库检索出乳腺癌发生、发展过程有意义的BAC克隆,然后利用CGAP数据库设计更有意义的BAC克隆。结果：获得1286条BAC克隆,可用于打印CCH微阵列,进行乳腺癌的检测。