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.     

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.     

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.     

Breaking the waves: improved detection of copy number variation from microarray-based comparative genomic hybridization

Background

Large-scale high throughput studies using microarray technology have established that copy number variation (CNV) throughout the genome is more frequent than previously thought. Such variation is known to play an important role in the presence and development of phenotypes such as HIV-1 infection and Alzheimer's disease. However, methods for analyzing the complex data produced and identifying regions of CNV are still being refined.

Results

We describe the presence of a genome-wide technical artifact, spatial autocorrelation or 'wave', which occurs in a large dataset used to determine the location of CNV across the genome. By removing this artifact we are able to obtain both a more biologically meaningful clustering of the data and an increase in the number of CNVs identified by current calling methods without a major increase in the number of false positives detected. Moreover, removing this artifact is critical for the development of a novel model-based CNV calling algorithm - CNVmix - that uses cross-sample information to identify regions of the genome where CNVs occur. For regions of CNV that are identified by both CNVmix and current methods, we demonstrate that CNVmix is better able to categorize samples into groups that represent copy number gains or losses.

Conclusion

Removing artifactual 'waves' (which appear to be a general feature of array comparative genomic hybridization (aCGH) datasets) and using cross-sample information when identifying CNVs enables more biological information to be extracted from aCGH experiments designed to investigate copy number variation in normal individuals.     

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.     

Copy number variation in Fayoumi and Leghorn chickens analyzed using array comparative genomic hybridization

Copy number variation refers to regions along chromosomes that harbor a type of structural variation, such as duplications or deletions. Copy number variants (CNVs) play a role in many important traits as well as in genetic diversity. Previous analyses of chickens using array comparative genomic hybridizations or single‐nucleotide polymorphism chip assays have been performed on various breeds and genetic lines to discover CNVs. In this study, we assessed individuals from two highly inbred (inbreeding coefficiency > 99.99%) lines, Leghorn G‐B2 and Fayoumi M15.2, to discover novel CNVs in chickens. These lines have been previously studied for disease resistance, and to our knowledge, this represents the first global assessment of CNVs in the Fayoumi breed. Genomic DNA from individuals was examined using the Agilent chicken 244 K comparative genomic hybridization array and quantitative PCR. We identified a total of 273 CNVs overall, with 112 CNVs being novel and not previously reported. Quantitative PCR using the standard curve method validated a subset of our array data. Through enrichment analysis of genes within CNV regions, we observed multiple chromosomes, terms and pathways that were significantly enriched, largely dealing with the major histocompatibility complex and immune responsiveness. Using an additional round of computational and statistical analysis with a different bioinformatic pipeline, we identified 43 CNVs among these as high‐confidence regions, 14 of which were found to be novel. We further compared and contrasted individuals of the two inbred lines to discover regions that have a significant difference in copy number between lines. A total of 40 regions had significant deletions or duplications between the lines. Gene Ontology analysis of genomic regions containing CNVs between lines also was performed. This between‐line candidate CNV list will be useful in studies with these two unique genetic lines, which may harbor variations that underlie quantitative trait loci for disease resistance and other important traits. Through the global discovery of novel CNVs in chicken, these data also provide resources for further genetic and functional genomics studies.     

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.     

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.     

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.     

Novel origins of copy number variation in the dog genome

ABSTRACT: BACKGROUND: Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. RESULTS: We use a stringent new method to identify a total of 430 high-confidence CNV loci, that range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. 98% of CNVs observed in each breed are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. CONCLUSIONS: A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution.     

The human genome puzzle - the role of copy number variation in somatic mosaicism

The discovery of copy number variations (CNV) in the human genome opened new perspectives in the study of the genetic causes of inherited disorders and the etiology of common diseases. Differently patterned instances of somatic mosaicism in CNV regions have been shown to be present in monozygotic twins and throughout different tissues within an individual. A single-cell-level investigation of CNV in different human cell types led us to uncover mitotically derived genomic mosaicism, which is stable in different cell types of one individual. A unique study of immortalized B-lymphoblastoid cell lines obtained with 20 year interval from the same two subjects shows that mitotic changes in CNV regions may happen early during embryonic development and seem to occur only once, as levels of mosaicism remained stable. This finding has the potential to change our concept of dynamic human genome variation. We propose that further genomic studies should focus on the single-cell level, to understand better the etiology and physiology of aging and diseases mediated by somatic variations.     

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 first comparative map of copy number variations in the sheep genome

We carried out a cross species cattle-sheep array comparative genome hybridization experiment to identify copy number variations (CNVs) in the sheep genome analysing ewes of Italian dairy or dual-purpose breeds (Bagnolese, Comisana, Laticauda, Massese, Sarda, and Valle del Belice) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. We identified 135 CNV regions (CNVRs; 24 reported in more than one animal) covering ~10.5 Mb of the virtual sheep genome referred to the bovine genome (0.398%) with a mean and a median equal to 77.6 and 55.9 kb, respectively. A comparative analysis between the identified sheep CNVRs and those reported in cattle and goat genomes indicated that overlaps between sheep and both other species CNVRs are highly significant (P<0.0001), suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Many sheep CNVRs include genes with important biological functions. Further studies are needed to evaluate their functional relevance.     

Clinical interpretation of copy number variants in the human genome

Molecular methods, by which copy number variants (CNVs) detection is available, have been gradually introduced into routine diagnostics over the last 15 years. Despite this, some CNVs continue to be a huge challenge when it comes to clinical interpretation. CNVs are an important source of normal and pathogenic variants, but, in many cases, their impact on human health depends on factors that are not yet known. Therefore, perception of their clinical consequences can change over time, as our knowledge grows. This review summarises guidelines that facilitate correct classification of identified changes and discusses difficulties with the interpretation of rare, small CNVs.     

Identification of germline genomic copy number variation in familial pancreatic cancer

Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10?% of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.     

Copy number variants (CNVs) in primate species using array-based comparative genomic hybridization

A substantial amount of genomic variation is now known to exist in humans and other primate species. Single nucleotide polymorphisms (SNPs) are thought to represent the vast majority of genomic differences among individuals in a given primate species and comprise about 0.1% of the genomes of two humans. However, recent studies have now shown that structural variation msay account for as much as 0.7% of the genomic differences in humans, of which copy number variants (CNVs) are the largest component. CNVs are segments of DNA that can range in size from hundreds of bases to millions of base pairs in length and have different number of copies between individuals. Recent technological advancements in array technologies led to genome-wide identification of CNVs and consequently revealed thousands of variable loci in humans, comprising as much as 12% of the human genome [A.J. Iafrate, L. Feuk, M.N. Rivera, M.L. Listewnik, P.K. Donahoe, Y. Qi, S.W. Scherer, C. Lee, Nat. Genet. 36 (2004) 949–951, [3]]. CNVs in humans have already been associated with susceptibility to certain complex diseases, dietary adaptation, and several neurological conditions. In addition, recent studies have shown that CNVs can be successfully implemented in population genetics research, providing important insights into human genetic variation. Nevertheless, the important role of CNVs in primate evolution and genetic diversity is still largely unknown. This article aims to outline the strengths and weaknesses of current comparative genomic hybridization array technologies that have been employed to detect CNV variation and the applications of these techniques to primate genetic research.     

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.     

A high-resolution map of segmental DNA copy number variation in the mouse genome

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.     

Identification of copy number variation hotspots in human populations

Copy number variants (CNVs) in the human genome contribute to both Mendelian and complex traits as well as to genomic plasticity in evolution. The investigation of mutational rates of CNVs is critical to understanding genomic instability and the etiology of the copy number variation (CNV)-related traits. However, the evaluation of the CNV mutation rate at the genome level poses an insurmountable practical challenge that requires large samples and accurate typing. In this study, we show that an approximate estimation of the CNV mutation rate could be achieved by using the phylogeny information of flanking SNPs. This allows a genome-wide comparison of mutation rates between CNVs with the use of vast, readily available data of SNP genotyping. A total of 4187 CNV regions (CNVRs) previously identified in HapMap populations were investigated in this study. We showed that the mutation rates for the majority of these CNVRs are at the order of 10⁻⁵ per generation, consistent with experimental observations at individual loci. Notably, the mutation rates of 104 (2.5%) CNVRs were estimated at the order of 10⁻³ per generation; therefore, they were identified as potential hotspots. Additional analyses revealed that genome architecture at CNV loci has a potential role in inciting mutational hotspots in the human genome. Interestingly, 49 (47%) CNV hotspots include human genes, some of which are known to be functional CNV loci (e.g., CNVs of C4 and β-defensin causing autoimmune diseases and CNVs of HYDIN with implication in control of cerebral cortex size), implicating the important role of CNV in human health and evolution, especially in common and complex diseases.