Emerging patterns of genome organization in Notopteridae species (Teleostei, Osteoglossiformes) as revealed by Zoo-FISH and Comparative Genomic Hybridization (CGH).

Notopteridae (Teleostei, Osteoglossiformes) represents an old fish lineage with ten currently recognized species distributed in African and Southeastern Asian rivers. Their karyotype structures and diploid numbers remained conserved over long evolutionary periods, since African and Asian lineages diverged approximately 120 Mya. However, a significant genetic diversity was already identified for these species using molecular data. Thus, why the evolutionary relationships within Notopteridae are so diverse at the genomic level but so conserved in terms of their karyotypes? In an attempt to develop a more comprehensive picture of the karyotype and genome evolution in Notopteridae, we performed comparative genomic hybridization (CGH) and cross-species (Zoo-FISH) whole chromosome painting experiments to explore chromosome-scale intergenomic divergence among seven notopterid species, collected in different African and Southeast Asian river basins. CGH demonstrated an advanced stage of sequence divergence among the species and Zoo-FISH experiments showed diffuse and limited homology on inter-generic level, showing a temporal reduction of evolutionarily conserved syntenic regions. The sharing of a conserved chromosomal region revealed by Zoo-FISH in these species provides perspectives that several other homologous syntenic regions have remained conserved among their genomes despite long temporal isolation. In summary, Notopteridae is an interesting model for tracking the chromosome evolution as it is (i) ancestral vertebrate group with Gondwanan distribution and (ii) an example of animal group exhibiting karyotype stasis. The present study brings new insights into degree of genome divergence vs. conservation at chromosomal and sub-chromosomal level in representative sampling of this group.

Malignant canine mammary tumours: Preliminary genomic insights using oligonucleotide array comparative genomic hybridisation analysis.

Neoplastic mammary disease in female dogs represents a major health concern for dog owners and veterinarians, but the genomic basis of the disease is poorly understood. In this study, we performed high resolution oligonucleotide array comparative genomic hybridisation (oaCGH) to assess genome wide DNA copy number changes in 10 malignant canine mammary tumours from seven female dogs, including multiple tumours collected at one time from each of three female dogs. In all but two tumours, genomic imbalances were detected, with losses being more common than gains. Canine chromosomes 9, 22, 26, 27, 34 and X were most frequently affected. Dissimilar oaCGH ratio profiles were observed in multiple tumours from the same dogs, providing preliminary evidence for probable independent pathogenesis. Analysis of adjacent samples of one tumour revealed regional differences in the number of genomic imbalances, suggesting heterogeneity within tumours.

Detection of Copy Number Imbalance in Canine Urothelial Carcinoma With Droplet Digital Polymerase Chain Reaction.

Urothelial carcinoma (UC) is the most common neoplasm of the canine urinary tract. Clinical presentation of UC is shared with several other, more common urinary tract disorders, and this often delays diagnosis of the UC. Definitive diagnosis of UC requires histopathologic examination of a biopsy specimen, but the cost and invasiveness for these diagnostic tests often result in most diagnoses being made on the basis of clinical findings, diagnostic imaging, and cytologic examination of urine sediment. Regardless of the diagnostic process used, most UCs currently are not diagnosed until they are at an advanced clinical stage and so are associated with poor prognosis. Improved methods for earlier and less invasive detection are needed. In a previous study, the authors demonstrated the presence of highly recurrent DNA copy number aberrations (CNAs) in canine UC and hypothesized that detection of these CNAs in tumor cells can be used as a molecular diagnostic for UC. In this study, a multiplexed droplet digital polymerase chain reaction (ddPCR) assay was detected to detect and quantify CNAs of specific regions of canine chromosomes 8, 13, 19, and 36. The assay was effective at differentiating 31 neoplastic and 25 nonneoplastic bladder tissues based on copy number, with 100% sensitivity and specificity in tissue samples. CNAs were also detected by ddPCR in 67% (12 of 18) of urine DNA specimens derived from UC patients. The findings show that ddPCR is a useful molecular technique to detect CNAs and may be used as a noninvasive molecular diagnostic test for canine UC.

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.

Global patterns of apparent copy number variation in birds revealed by cross-species comparative genomic hybridization.

There is a growing interest in copy number variation (CNV) and the recognition of its importance in phenotype, disease, adaptation and speciation. CNV data is usually ascertained by array-CGH within-species, but similar inter-species comparisons have also been made in primates, mice and domestic mammals. Here, we conducted a broad appraisal of putative cross-species CNVs in birds, 16 species in all, using the standard array-CGH approach. Using a chicken oligonucleotide microarray, we detected 790 apparent CNVs within 135 unique regions and developed a bioinformatic tool 'CNV Analyser' for analysing and visualising cross-species data sets. We successfully addressed four hypotheses as follows: (a) Cross-species CNVs (compared to chicken) are, as suggested from preliminary evidence, smaller and fewer in number than in mammals; this 'dogma' was rejected in the light of the new evidence. (b) CNVs in birds are likely to have a functional effect through an association with genes; a large proportion of detected regions (70 %) were indeed associated with genes (suggesting functional significance), however, not necessarily more so than in mammals. (c) There are more CNVs in birds with more rearranged karyotypes; this hypothesis was rejected. Indeed, Falco species contained fewer than most with relatively standard (chicken-like) karyotypes. (d) There are more CNVs per megabase on micro-chromosomes than macrochromosomes; this hypothesis was accepted. Indeed, in species with rearranged karyotypes characterised by chromosomal fusions, the fused former microchromosomes still 'behaved' as though they were their microchromosomal ancestors. Gene ontology analysis of CNVRs revealed enrichment in immune response and antigen presentation genes and five CNVRs were perfectly correlated with the unique loss of sexual dichromatism in one Galliformes species.

Molecular characterization of canine BCR-ABL-positive chronic myelomonocytic leukemia before and after chemotherapy.

Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High-resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region-Abelson murine leukemia viral oncogene homolog (BCR-ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR-ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.

Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

Identification of genome-wide copy number variations among diverse pig breeds by array CGH.

BACKGROUND: Recent studies have shown that copy number variation (CNV) in mammalian genomes contributes to phenotypic diversity, including health and disease status. In domestic pigs, CNV has been catalogued by several reports, but the extent of CNV and the phenotypic effects are far from clear. The goal of this study was to identify CNV regions (CNVRs) in pigs based on array comparative genome hybridization (aCGH). RESULTS: Here a custom-made tiling oligo-nucleotide array was used with a median probe spacing of 2506 bp for screening 12 pigs including 3 Chinese native pigs (one Chinese Erhualian, one Tongcheng and one Yangxin pig), 5 European pigs (one Large White, one Pietrain, one White Duroc and two Landrace pigs), 2 synthetic pigs (Chinese new line DIV pigs) and 2 crossbred pigs (Landrace × DIV pigs) with a Duroc pig as the reference. Two hundred and fifty-nine CNVRs across chromosomes 1-18 and X were identified, with an average size of 65.07 kb and a median size of 98.74 kb, covering 16.85 Mb or 0.74% of the whole genome. Concerning copy number status, 93 (35.91%) CNVRs were called as gains, 140 (54.05%) were called as losses and the remaining 26 (10.04%) were called as both gains and losses. Of all detected CNVRs, 171 (66.02%) and 34 (13.13%) CNVRs directly overlapped with Sus scrofa duplicated sequences and pig QTLs, respectively. The CNVRs encompassed 372 full length Ensembl transcripts. Two CNVRs identified by aCGH were validated using real-time quantitative PCR (qPCR). CONCLUSIONS: Using 720 K array CGH (aCGH) we described a map of porcine CNVs which facilitated the identification of structural variations for important phenotypes and the assessment of the genetic diversity of pigs.

Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array.

BACKGROUND: Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases. RESULTS: In this study using the high density BovineHD SNP array, we performed high resolution CNV analyses on both Btau_4.0 and UMD3.1 with 674 animals of 27 cattle breeds. We first compared CNV results derived from these two different SNP array platforms on Btau_4.0. With two thirds of the animals shared between studies, on Btau_4.0 we identified 3,346 candidate CNV regions representing 142.7 megabases (~4.70%) of the genome. With a similar total length but 5 times more event counts, the average CNVR length of current Btau_4.0 dataset is significantly shorter than the previous one (42.7 kb vs. 205 kb). Although subsets of these two results overlapped, 64% (91.6 megabases) of current dataset was not present in the previous study. We also performed similar analyses on UMD3.1 using these BovineHD SNP array results. Approximately 50% more and 20% longer CNVs were called on UMD3.1 as compared to those on Btau_4.0. However, a comparable result of CNVRs (3,438 regions with a total length 146.9 megabases) was obtained. We suspect that these results are due to the UMD3.1 assembly's efforts of placing unplaced contigs and removing unmerged alleles. Selected CNVs were further experimentally validated, achieving a 73% PCR validation rate, which is considerably higher than the previous validation rate. About 20-45% of CNV regions overlapped with cattle RefSeq genes and Ensembl genes. Panther and IPA analyses indicated that these genes provide a wide spectrum of biological processes involving immune system, lipid metabolism, cell, organism and system development. CONCLUSION: We present a comprehensive result of cattle CNVs at a higher resolution and sensitivity. We identified over 3,000 candidate CNV regions on both Btau_4.0 and UMD3.1, further compared current datasets with previous results, and examined the impacts of genome assemblies on CNV calling.

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

OBJECTIVE: To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. SAMPLE: Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. PROCEDURES: Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. RESULTS: LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. CONCLUSIONS AND CLINICAL RELEVANCE: Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

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.

Genetic characterization of dogs via chromosomal analysis and array-based comparative genomic hybridization (aCGH).

The results of cytogenetic and molecular cytogenetic investigations revealed similarities in genetic background and biological behaviour between tumours and genetic diseases of humans and dogs. These findings classify the dog a good and accepted model for human cancers such as osteosarcomas, mammary carcinomas, oral melanomas and others. With the appearance of new studies and advances in canine genome sequencing, the number of known homologies in diseases between these species raised and still is expected to increase. In this context, array-based comparative genomic hybridization (aCGH) provides a novel tool to rapidly characterize numerical aberrations in canine tumours or to detect copy number aberrations between different breeds. As it is possible to spot probes covering the whole genome on each chip to discover copy number aberrations of all chromosomes simultaneously, this method is time-saving and cost-effective - considering the relation of costs and the amount of data obtained. Complemented with traditional methods like karyotyping and fluorescence in situ hybridization (FISH) analyses, the aCGH is able to provide new insights into the underlying causes of canine carcinogenesis.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.