Cytogenetic analysis from DNA by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.     

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.     

Karyotyping of comparative genomic hybridization human metaphases by using support vector machines.

BACKGROUND: Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method for detecting chromosomal imbalance. Karyotyping of human metaphases is an important step to assign each chromosome to one of 23 or 24 classes (22 autosomes and two sex chromosomes). Automatic karyotyping in CGH analysis is needed. However, conventional karyotyping approaches based on DAPI images require complex image enhancement procedures. METHODS: This paper proposes a simple feature extraction method, one that generates density profiles from original true color CGH images and uses normalized profiles as feature vectors without quantization. A classifier is developed by using support vector machine (SVM). It has good generalization ability and needs only limited training samples. RESULTS: Experiment results show that the feature extraction method of using color information in CGH images can improve greatly the classification success rate. The SVM classifier is able to acquire knowledge about human chromosomes from relatively few samples and has good generalization ability. A success rate of moe than 90% has been achieved and the time for training and testing is very short. CONCLUSIONS: The feature extraction method proposed here and the SVM-based classifier offer a promising computerized intelligent system for automatic karyotyping of CGH human chromosomes.     

Karyotyping of comparative genomic hybridization human metaphases using kernel nearest-neighbor algorithm

BACKGROUND: Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method that detects chromosomal imbalances. Automatic karyotyping is an important step in CGH analysis because the precise position of the chromosome abnormality must be located and manual karyotyping is tedious and time-consuming. In the past, computer-aided karyotyping was done by using the 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)-inverse images, which required complex image enhancement procedures. METHODS: An innovative method, kernel nearest-neighbor (K-NN) algorithm, is proposed to accomplish automatic karyotyping. The algorithm is an application of the "kernel approach," which offers an alternative solution to linear learning machines by mapping data into a high dimensional feature space. By implicitly calculating Euclidean or Mahalanobis distance in a high dimensional image feature space, two kinds of K-NN algorithms are obtained. New feature extraction methods concerning multicolor information in CGH images are used for the first time. RESULTS: Experiment results show that the feature extraction method of using multicolor information in CGH images improves greatly the classification success rate. A high success rate of about 91.5% has been achieved, which shows that the K-NN classifier efficiently accomplishes automatic chromosome classification from relatively few samples. CONCLUSIONS: The feature extraction method proposed here and K-NN classifiers offer a promising computerized intelligent system for automatic karyotyping of CGH human chromosomes.     

Detecting copy number variation in the human genome using comparative genomic hybridization

Among human beings, it was once estimated that our genomes were 99.9% genetically identical. While this high level of genetic similarity helps to define us as a species, it is our genetic variation that contributes to our phenotypic diversity. As genomic technologies evolve to provide genome-wide analyses at higher resolution, we are beginning to appreciate that the human genome has a lot more variation than was once thought. Array-based comparative genomic hybridization (CGH) is one of these technologies that has recently revealed a newly appreciated type of genetic variation: copy number variation, in which thousands of regions of the human genome are now known to be variable in number between individuals. Some of these copy number variable regions have already been shown to predispose to certain common diseases, and others may ultimately have a significant impact on how each of us reacts to certain foods (e.g., allergic reactions), medications (e.g., pharmacogenomics), microscopic infections (i.e., immunity), and other aspects of our ever-changing environment.     

Denoising array-based comparative genomic hybridization data using wavelets

Array-based comparative genomic hybridization (array-CGH) provides a high-throughput, high-resolution method to measure relative changes in DNA copy number simultaneously at thousands of genomic loci. Typically, these measurements are reported and displayed linearly on chromosome maps, and gains and losses are detected as deviations from normal diploid cells. We propose that one may consider denoising the data to uncover the true copy number changes before drawing inferences on the patterns of aberrations in the samples. Nonparametric techniques are particularly suitable for data denoising as they do not impose a parametric model in finding structures in the data. In this paper, we employ wavelets to denoise the data as wavelets have sound theoretical properties and a fast computational algorithm, and are particularly well suited for handling the abrupt changes seen in array-CGH data. A simulation study shows that denoising data prior to testing can achieve greater power in detecting the aberrant spot than using the raw data without denoising. Finally, we illustrate the method on two array-CGH data sets.     

Leptospire genomic diversity revealed by microarray-based comparative genomic hybridization

Comparative genomic hybridization was used to compare genetic diversity of five strains of Leptospira (Leptospira interrogans serovars Bratislava, Canicola, and Hebdomadis and Leptospira kirschneri serovars Cynopteri and Grippotyphosa). The array was designed based on two available sequenced Leptospira reference genomes, those of L. interrogans serovar Copenhageni and L. interrogans serovar Lai. A comparison of genetic contents showed that L. interrogans serovar Bratislava was closest to the reference genomes while L. kirschneri serovar Grippotyphosa had the least similarity to the reference genomes. Cluster analysis indicated that L. interrogans serovars Bratislava and Hebdomadis clustered together first, followed by L. interrogans serovar Canicola, before the two L. kirschneri strains. Confirmed/potential virulence factors identified in previous research were also detected in the tested strains.     

Whole-chromosome aneuploidy analysis in human oocytes: focus on comparative genomic hybridization

The study of aneuploidy in human oocytes, discarded from IVF cycles, has provided a better understanding of the incidence of aneuploidy of female origin and the responsible mechanisms. Comparative genomic hybridization (CGH) is an established technique that allows for the detection of aneuploidy in all chromosomes avoiding artifactual chromosome losses. In this review, results obtained using CGH in single cells (1PB and/or MII oocytes) are included. The results of oocyte aneuploidy rates obtained by CGH from discarded oocytes of IVF patients and of oocyte donors are summarized. Moreover, the mechanisms involved in the aneuploid events, e.g. whether alterations occurred due to first meiotic errors or germ-line mitotic errors are also discussed. Finally, the incidence of aneuploid oocyte production due to first meiotic errors and germ-line mitotic errors observed in oocytes coming from IVF patients and IVF oocyte donors was assessed.     

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.     

High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.     

Analysis of genetic aberrations in uterine adenomyosis using comparative genomic hybridization

OBJECTIVE: To use comparative genomic hybridization (CGH) to analyzefrozen tissuesfrom adenomyosis cases to discover and map genomic regions for chromosomal gains and losses. STUDY DESIGN: In a retrospective study, upper and lower threshold values of 1.20 and 0.80, respectively, were used to define positive findings. RESULTS: No positive recurrent gene copy number alterations were detected in the 25 cases of pathologically proven adenomyosis. CONCLUSION: Although CGH is extremely useful in investigating candidate genes in the development of adenomyosis, CGH was not useful in this study. Genetic changes might be indeed extremely rare in adenomyosis, or CGH was not sensitive enough to detect candidate genes.     

Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs.     

Stochastic segmentation models for array-based comparative genomic hybridization data analysis

Array-based comparative genomic hybridization (array-CGH) is a high throughput, high resolution technique for studying the genetics of cancer. Analysis of array-CGH data typically involves estimation of the underlying chromosome copy numbers from the log fluorescence ratios and segmenting the chromosome into regions with the same copy number at each location. We propose for the analysis of array-CGH data, a new stochastic segmentation model and an associated estimation procedure that has attractive statistical and computational properties. An important benefit of this Bayesian segmentation model is that it yields explicit formulas for posterior means, which can be used to estimate the signal directly without performing segmentation. Other quantities relating to the posterior distribution that are useful for providing confidence assessments of any given segmentation can also be estimated by using our method. We propose an approximation method whose computation time is linear in sequence length which makes our method practically applicable to the new higher density arrays. Simulation studies and applications to real array-CGH data illustrate the advantages of the proposed approach.     

Analysis of wilms tumors using SNP mapping array-based comparative genomic hybridization

Wilms tumor (WT) has been a model to study kidney embryogenesis and tumorigenesis and, although associated with hereditary, cancer predisposition syndromes, the majority of tumors occur sporadically. To analyze genetic changes in WT we have defined copy number changes and loss of heterozygosity in 56 Wilms tumors using high resolution oligonucleotide arrays at a average resolution of ~12 Kb. Consistent deletions were seen on chromosomes 1p, 4q, 7p, 9q, 11p, 11q, 14q, 16q, and 21q. High frequency gains were seen for 1q and lower frequency gains were seen on 7q and chromosomes 8, 12 and 18. The high resolution provided by the SNP mapping arrays has defined minimal regions of deletion for many of these LOH events. Analysis of CNAs by tumor stage show relatively stable karyotypes in stage 1 tumors and more complex aCGH profiles in tumors from stages 3-5.     

Genetic changes in human fetuses from spontaneous abortion after in vitro fertilization detected by comparative genomic hybridization

The in vitro fertilization (IVF) technique is becoming a very important approach for infertile disease therapy, but approximately 30% of pregnancies are spontaneously aborted in the first trimester. It is believed that chromosomal abnormality is the major reason for early spontaneous abortion. Although some reports have mentioned cytogenetic changes in spontaneously aborted embryos after IVF, little is known about the comprehensive cytogenetic alterations in these aborted embryos. Here we use the comparative genomic hybridization (CGH) technique to analyze the genetic alterations in 41 spontaneously aborted human specimens after IVF. In this study, 25 of 41 cases (61%) showed chromosomal changes. Among them, autosomes and sex chromosomes were involved in 16 and 11 cases, respectively. Several nonrandom chromosomal changes were identified, including loss of one sex chromosome (six cases) and gains of 22 (four cases), Y (four cases), 21 (three cases), 4 (two cases), and 13 (two cases). Our data support the opinion that chromosome abnormality is one of the major causes of early spontaneous abortion after IVF. The association between chromosome changes in these spontaneously aborted fetuses and maternal age, infertility patterns, infertility causes, and IVF patterns (routine IVF and other methods, including intracytoplasmic sperm injection, egg donation, and embryo donation) were also studied. No significant correlation was found.     

Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.     

Survey of genomic diversity among Enterococcus faecalis strains by microarray-based comparative genomic hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the "test" strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies

Single cell comparative genomic hybridization (CGH) was employed to extensively investigate 24 unfertilized or in vitromatured meiosis II oocytes and their corresponding first polar bodies (PBs), to determine how and whether all 23 chromosomes participate in female meiosis I errors and to accurately estimate the aneuploidy rate in the examined cells. Results were obtained for 15 oocytes and 16 PBs, representing 23 eggs (MII oocyte-PB complexes) donated from 15 patients (average age 32.2 years). Abnormalities were detected in ten eggs, giving an overall aneuploidy rate of 43.5%. In all, fourteen anomalies were scored, with the fertilized oocyte being at risk of monosomy in eight cases and at risk of trisomy in six; chromosomes of various sizes participated. CGH was able to give a comprehensive aneuploidy rate, as both absence of chromosomal material and the presence of extra copies were accurately scored. The aneuploidy mechanisms determined were: classical whole univalent non-disjunction; chromatid predivision prior to anaphase I, leading to metaphase II imbalance. There was also evidence of germinal mosaicism for a trisomic cell line. Three patients appeared to be predisposed to meiosis I errors, based on the presence of either multiple abnormalities in one or more of their examined cells, or of the same type of abnormality in all of their cells. Exclusion of these susceptible patients reduces the aneuploidy rate to 20%. Various hypotheses are put forward to explain these observations in order to stimulate research into the complex nature of female meiotic regulation.