Identification of large-scale human-specific copy number differences by inter-species array comparative genomic hybridization

Copy number differences (CNDs), and the concomitant differences in gene number, have contributed significantly to the genomic divergence between humans and other primates. To assess its relative importance, the genomes of human, common chimpanzee, bonobo, gorilla, orangutan and macaque were compared by comparative genomic hybridization using a high-resolution human BAC array (aCGH). In an attempt to avoid potential interference from frequent intra-species polymorphism, pooled DNA samples were used from each species. A total of 322 sites of large-scale inter-species CND were identified. Most CNDs were lineage-specific but frequencies differed considerably between the lineages; the highest CND frequency among hominoids was observed in gorilla. The conserved nature of the orangutan genome has already been noted by karyotypic studies and our findings suggest that this degree of conservation may extend to the sub-microscopic level. Of the 322 CND sites identified, 14 human lineage-specific gains were observed. Most of these human-specific copy number gains span regions previously identified as segmental duplications (SDs) and our study demonstrates that SDs are major sites of CND between the genomes of humans and other primates. Four of the human-specific CNDs detected by aCGH map close to the breakpoints of human-specific karyotypic changes [e.g., the human-specific inversion of chromosome 1 and the polymorphic inversion inv(2)(p11.2q13)], suggesting that human-specific duplications may have predisposed to chromosomal rearrangement. The association of human-specific copy number gains with chromosomal breakpoints emphasizes their potential importance in mediating karyotypic evolution as well as in promoting human genomic diversity. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Characterization of breast cancer by array comparative genomic hybridization.

Cancer progression is due to the accumulation of recurrent genomic alterations that induce growth advantage and clonal expansion. Most of these genomic changes can be detected using the array comparative genomic hybridization (CGH) technique. The accurate classification of these genomic alterations is expected to have an important impact on translational and basic research. Here we review recent advances in CGH technology used in the characterization of different features of breast cancer. First, we present bioinformatics methods that have been developed for the analysis of CGH arrays; next, we discuss the use of array CGH technology to classify tumor stages and to identify and stratify subgroups of patients with different prognoses and clinical behaviors. We finish our review with a discussion of how CGH arrays are being used to identify oncogenes, tumor suppressor genes, and breast cancer susceptibility genes.     

Array-based comparative genomic hybridization and copy number variation in cancer research

Array-based comparative genomic hybridization (aCGH) is a molecular cytogenetic technique used in detecting and mapping DNA copy number alterations. aCGH is able to interrogate the entire genome at a previously unattainable, high resolution and has directly led to the recent appreciation of a novel class of genomic variation: copy number variation (CNV) in mammalian genomes. All forms of DNA variation/polymorphism are important for studying the basis of phenotypic diversity among individuals. CNV research is still at its infancy, requiring careful collation and annotation of accumulating CNV data that will undoubtedly be useful for accurate interpretation of genomic imbalances identified during cancer research.     

DNA copy number changes in human malignant fibrous histiocytomas by array comparative genomic hybridisation

Background

Malignant fibrous histiocytomas (MFHs), or undifferentiated pleomorphic sarcomas, are in general high-grade tumours with extensive chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, as well as novel gene targets of potential importance for MFH development and/or progression, we have analysed DNA copy number changes in 33 MFHs using microarray-based comparative genomic hybridisation (array CGH).

Principal findings

In general, the tumours showed numerous gains and losses of large chromosomal regions. The most frequent minimal recurrent regions of gain were 1p33-p32.3, 1p31.3-p31.2 and 1p21.3 (all gained in 58% of the samples), as well as 1q21.2-q21.3 and 20q13.2 (both 55%). The most frequent minimal recurrent regions of loss were 10q25.3-q26.11, 13q13.3-q14.2 and 13q14.3-q21.1 (all lost in 64% of the samples), as well as 2q36.3-q37.2 (61%), 1q41 (55%) and 16q12.1-q12.2 (52%). Statistical analyses revealed that gain of 1p33-p32.3 and 1p21.3 was significantly associated with better patient survival (P = 0.021 and 0.046, respectively). Comparison with similar array CGH data from 44 leiomyosarcomas identified seven chromosomal regions; 1p36.32-p35.2, 1p21.3-p21.1, 1q32.1-q42.13, 2q14.1-q22.2, 4q33-q34.3, 6p25.1-p21.32 and 7p22.3-p13, which were significantly different in copy number between the MFHs and leiomyosarcomas.

Conclusions

A number of recurrent regions of gain and loss have been identified, some of which were associated with better patient survival. Several specific chromosomal regions with significant differences in copy number between MFHs and leiomyosarcomas were identified, and these aberrations may be used as additional tools for the differential diagnosis of MFHs and leiomyosarcomas.     

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.     

Gene copy number changes in dermatofibrosarcoma protuberans - a fine-resolution study using array comparative genomic hybridization

Dermatofibrosarcoma protuberans (DFSP) is a rare, slow-growing, low-grade dermal tumor. Cytogenetic and FISH studies have revealed that the chromosomal rearrangements characteristic of DFSP tumors involve both translocations and the formation of a supernumerary ring derived from chromosomes 17 and 22. The t(17;22) (q22;q13.1) translocation generates a gene fusion between COL1A1 and PDGFB, which serves as a diagnostic marker of DFSP. In the present study we performed array-CGH (aCGH) analysis on ten DFSP tumors. The COL1A1 region at 17q was gained in 71% (5/7) of the samples and the PDGFB region at 22q was gained in 43% (3/7) of the individual samples. In addition to the 17q and 22q gains, altogether 17 minimal common regions of gain and one region of loss were detected.     

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.     

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.     

Copy number imbalances detected with a BAC-based array comparative genomic hybridization platform in congenital diaphragmatic hernia fetuses

Congenital diaphragmatic hernia (CDH) is a phenotypically and genetically heterogeneous disorder, with a complex inheritance pattern. Structural abnormalities of almost all chromosomes have been described in association with CDH. We made a molecular analysis through array comparative genomic hybridization (array CGH) of a group of fetuses with prenatal ultrasound diagnosis of CDH and normal G-banded karyotypes. A whole genome BAC-array CGH, composed of approximately 5000 BAC clones, was carried out on blood samples from fetuses with prenatal ultrasound diagnosis of CDH and a normal karyotype (500-band level). All potential cytogenetic alterations detected on the arrays were reported. The array CGH analysis showed copy number gains and losses in 10 of 12 cases. Eighty-five clones showed genomic imbalances, and 29 clones displayed described copy number variations. We identified a recurrent gain in 17q12 in two of 12 cases, which has not been previously described. Our results may contribute to determining the effectiveness and applicability of array CGH for prenatal diagnosis purposes, and also to elucidate the submicroscopic genomic instability of CDH fetuses.     

DNA copy number evolution in Drosophila cell lines

Background

Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles.

Results

Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167).

Conclusion

Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/gb-2014-15-8-r70) contains supplementary material, which is available to authorized users.     

The most common chromosome aberration detected by high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma

We analyzed genetic changes in condylomas (four cases), vulvar intraepithelial neoplasia I-III (VIN I-III, eleven cases), and primary vulvar squamous cell carcinomas (VSCC, ten cases) by high-resolution comparative genomic hybridization (HR-CGH) and flowcytometry. All samples were also human papilloma virus (HPV)-genotyped. Gain of chromosome 1, the aberration most often seen in VIN III (67%), was not seen in HPV-positive or -negative VSCCs (0%). Both VIN III and VSCC frequently showed gain of 3q (56 and 70%, respectively). The VIN III samples often demonstrated gain of 20q (56%) and 20p (44%), and the VSCC samples gain of 8q (60%), loss of 3p (50%), and 8p (40%). None of the four most frequent changes in the VSCC samples occurred exclusively in the HPV-positive or -negative samples. As expected, we did not find any cytogenetic changes in condylomas and nearly any changes in VIN I-II.     

Significant involvement of chromosome 13q deletions in progression of larynx cancer, detected by comparative genomic hybridization

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumours with various clinical characteristics. These tumours generally exhibit complex karyotypes. Few studies of genomic imbalances have been performed exclusively in subgroups of larynx cancer samples at different stages of the disease. In the present study, chromosomal gains and losses were investigated in 52 larynx tumours, by using comparative genomic hybridization (CGH). The mean number of observed alterations was 37.7 per tumour. The most common sites of losses were 1p, 13q, Xp, and the most common gains were located in 1p, 9q, 16q. The overall number of gains was negatively associated with cancer grading. G1 tumours were also characterized by a higher frequency of deletions in 13q32 and amplifications in 1q23, than tumours in other grades (p < 0.05). The frequency of losses of 13q22 also positively associated with tumour size. There was no association between the frequency of losses in 13q and the presence of lymph node metastases at the time of diagnosis. Another statistically significant association was observed for gains at 1q22-23 and tumour size (p < 0.01). No statistically significant difference in the frequency of most common imbalances was detected between primary tumours with lymph node metastases and those without metastases. In conclusion, we discovered a significant involvement of 13q deletions in the progression of larynx cancer. All the other significant changes observed in the present study were reported previously as being important for HNSCC progression. It seems that multiple genes are disrupted in the process of neoplastic transformation in the larynx, and the networks of events remain to be elucidated.     

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.     

Fuzzy methods for the detection of copy number variations in comparative genomic hybridization arrays

Genomic copy number variations (CNVs) are considered as a significant source of genetic diversity and widely involved in gene expression and regulatory mechanism, genetic disorders and disease risk, susceptibility to certain diseases and conditions, and resistance to medical drugs. Many studies have targeted the identification, profiling, analysis, and associations of genetic CNVs. We propose herein two new fuzzy methods, taht is, one based on the fuzzy inference from the pre-processed input, and another based on fuzzy C-means clustering. Our solutions present a higher true positive rate and a lower false negative with no false positive, efficient performance and consumption of least resources.     

Abnormal chromosome 8 copy number in cytological smears from breast carcinomas detected by means of fluorescence in situ hybridization (FISH)

Numerical change in chromosome 8 is an acquired abnormality associated with high clinical stage and may be involved in the conversion of carcinoma in situ in the breast to invasive carcinoma. Fine needle aspiration smears from 53 cases of breast carcinoma were hybridized with centromeric probes for chromosome 8 and the X chromosome. Thirty-eight cases revealed chromosome 8 copy gain. Of the 45 grade II and III tumours, 28 showed polysomy (>3 signals) and six showed trisomy. Of the eight grade I tumours, four were trisomic, none were polysomic. There were only two cases of chromosome 8 copy loss (one each of grade I and III). X chromosome polysomy was also a frequent finding although the signal counts were similar to those for chromosome 8 in only a few cases. Chromosome 8 polysomy occurs frequently in breast carcinoma and high copy number (>3) is associated with high malignancy grade.     

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.     

Exploring copy number variation in the rabbit (Oryctolagus cuniculus) genome by array comparative genome hybridization

The European rabbit (Oryctolagus cuniculus) is relevant in a large spectrum of fields: it is a livestock, a pet, a biomedical model and a biotechnology tool, a wild resource and a pest. The sequencing of the rabbit genome has opened new perspectives to study this lagomorph at the genome level. We herein investigated for the first time the O. cuniculus genome by array comparative genome hybridization (aCGH) and established a first copy number variation (CNV) genome map in this species comprising 155 copy number variation regions (CNVRs; 95 gains, 59 losses, 1 with both gain and loss) covering ~0.3% of the OryCun2.0 version. About 50% of the 155 CNVRs identified spanned 139 different protein coding genes, 110 genes of which were annotated or partially annotated (including Major Histocompatibility Complex genes) with 277 different gene ontology terms. Many rabbit CNVRs might have a functional relevance that should be further investigated.