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PubMed2005

Comparative genomic hybridization.

Altering DNA copy number is one of the many ways that gene expression and function may be modified. Some variations are found among normal individuals ( 14, 35, 103 ), others occur in the course of normal processes in some species ( 33 ), and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur prior to or shortly after fertilization, whereas DNA dosage alterations that occur in somatic cells are frequent contributors to cancer. Detecting these aberrations, and interpreting them within the context of broader knowledge, facilitates identification of critical genes and pathways involved in biological processes and diseases, and provides clinically relevant information. Over the past several years array comparative genomic hybridization (array CGH) has demonstrated its value for analyzing DNA copy number variations. In this review we discuss the state of the art of array CGH and its applications in medical genetics and cancer, emphasizing general concepts rather than specific results.

Chromosome Aberrations↗
PubMed2001

Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.

Animals↗
PubMed1996

[CGH (comparative genomic hybridization)].

Comparative Genomic Hybridization (CGH) is a powerful new method which allows genome-wide mapping of regions with DNA sequence copy number changes (both increases and decreases) in a single experiment without previous knowledge of the locations of the regions of abnormality. CGH is based on in situ hybridization of differentially labeled total genomic tumor DNA and normal DNA to normal human metaphase chromosomes. After hybridization copy number variations among the sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target chromosomes. Many previously unknown chromosomal regions with relative copy number changes have been detected in various tumors by CGH. Some changes have been identified as genetic markers associated with biological and clinico-pathological characteristics (i.e., histopathological grade, and clinical outcome). We review the published CGH articles and discuss briefly on current progress in CGH analysis to ovarian and uterine cervical cancer in our laboratory.

DNA, Neoplasm↗
PubMed1995

Comparative genomic hybridization analysis of human sarcomas: I. Occurrence of genomic imbalances and identification of a novel major amplicon at 1q21-q22 in soft tissue sarcomas.

Comparative genomic hybridization (CGH) was recently developed as a tool to survey entire genomes for variations in DNA sequence copy numbers. We have applied this technique to detect and map amplified regions in 54 soft tissue sarcomas. Aberrations were detected by visual analysis of hybridizations or contrast-enhanced digital images, followed by quantitative digital ratio imaging of the aberrant chromosomes. Several tumors showed increased DNA sequence copy number at 12q14, as expected. However, CGH analysis detected amplification of 12q14 also in some tumors where neither MDM2 nor CDK4 was amplified, suggesting that another as yet unknown gene(s) may drive amplification of this region in sarcomas. Furthermore, a novel recurring amplicon was detected at 1q21-q22. DNA amplifications coinciding with this segment were as frequent as those observed for 12q14, indicating that 1q21-q22-linked gene(s) may also play an important role in the development and/or progression of human soft tissue sarcomas.

Animals↗
PubMed1976

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.

Animals↗
PubMed2026

Urinary Small Extracellular Vesicle DNA as a Biomarker for the Non-Invasive Diagnosis of Bladder Cancer.

Existing diagnostic technologies for bladder cancer (BC) suffer from low sensitivity, low specificity, or a lack of validation. Therefore, validated, non-invasive diagnostic biomarkers with high sensitivity and specificity for early detection of BC are needed to complement and improve upon the limitations of existing diagnostic methods. We used low-pass whole genome sequencing (LP-WGS) technology to detect copy number variations (CNVs) in small extracellular vesicle (sEV) DNA isolated from urine samples of patients. Based on these results, we constructed and validated a diagnostic model to differentiate between benign and malignant bladder lesions. We conducted a receiver operating characteristic analysis and calculated the area under the curve (AUC) to evaluate the performance of the diagnostic model. The urine sEV-DNA LP-WGS data revealed CNV differences between benign and malignant samples. The diagnostic model achieved an AUC of 0.953, a sensitivity of 86.7%, and a specificity of 100% in the training cohort and an AUC of 0.985, a sensitivity of 90%, and a specificity of 100% in the validation cohort. Even at the lowest coverage depth of 0.01X, the performance of the diagnostic model remained relatively robust. Notably, the performance of this diagnostic model surpassed that of the biomarker neuron-specific enolase (sensitivity: 85.7% vs. 64.3%; specificity: 100% vs. 87.5%) and urinary cytology (sensitivity: 100% vs. 66.7%; specificity: 100% vs. 94.1%). Our study demonstrates that urine sEV-DNA exhibits high discriminatory power in distinguishing between benign and malignant bladder lesions, making it a promising tool for auxiliary diagnosis of BC.

Humans↗
PubMed2026

Age-associated epigenomic heterogeneity in papillary tumors of the pineal region: a multicenter YoungNOA investigation.

BACKGROUND: Papillary tumors of the pineal region (PTPR) are rare CNS neoplasms with adult and pediatric presentations, but whether age defines distinct molecular biology is unclear. METHODS: We assembled a multicenter retrospective cohort of 86 histologically confirmed PTPR with genome-wide DNA methylation data, comprising 62 adult and 24 pediatric tumors. Molecular subgroup, array platform, sex, and tumor purity were incorporated into multivariable models. Analyses included DNA methylation class assignment, differential methylation, copy-number variation (CNV), epigenetic mitotic-clock scores, methylation-based tumor microenvironment deconvolution, and descriptive survival evaluation. RESULTS: Adult and pediatric tumors mapped within the established PTPR-A and PTPR-B methylation framework rather than forming age-defined methylation classes. Pediatric tumors were enriched for PTPR-B (22 of 24 tumors [91.7%]) compared with adult tumors (39 of 62 [62.9%]). After adjustment for methylation-based subgroup as well as technical and biological covariates, 2,923 CpG probes were associated with age at a false discovery rate (FDR) threshold below 0.05, and 530 also met the prespecified effect-size threshold. Global methylation summaries were similar between age groups. CNV patterns were dominated by molecular subgroup; adjusted genomic CNV load was not independently associated with pediatric age. In contrast, epiTOC2 intrinsic rate score and the methylation signature represented by the first principal component (PC1) showed age-associated effects independent of molecular subgroup. Methylation-based deconvolution suggested a limited microenvironmental signal, with neutrophil fraction showing the most consistent adjusted association. CONCLUSIONS: Adult and pediatric PTPR share the established PTPR-A/PTPR-B framework. Pediatric tumors, particularly within PTPR-B, showed age-associated DNA methylation differences and higher epigenetic mitotic-clock (epiTOC2) scores in this retrospective cohort. These tissue-level associations do not establish clinical risk or treatment implications and require prospective clinical annotation and orthogonal validation.

Humans↗
PubMed2006

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.

Animals↗
PubMed1986

Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125.

Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.

Animals↗
PubMed1994

Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.

Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.

Chromosome Aberrations↗
PubMed2004

Chromosomal polymorphism, gene synteny and genome size in T. cruzi I and T. cruzi II groups.

Pulsed-field gel electrophoresis and DNA hybridization were used to establish and compare some parameters of the molecular karyotype of nine stocks classified into Trypanosoma cruzi I and T. cruzi II groups. The isolates showed a variable number of chromosomal bands (17-22) comprised between 0.4 and 3.3 Mbp. The total number of chromosomes and the genome size were estimated based on the fluorescence intensity of SYBR Green I-stained chromosomal bands. Differences in the length of the telomeric regions among the stocks and between chromosomes of the same stock were observed. No correlation was found between the length of the telomeric region and the group to which the isolate belongs. Hybridization of 54 genetic markers revealed extensive chromosome size polymorphism. Nevertheless, the most represented pattern was the hybridization of the probes in larger chromosomes in stocks of T. cruzi II as compared to T. cruzi I. Eight putative syntenic groups, encompassing 29 non-redundant genetic markers and distributed in 11 CL Brener chromosomal bands were disclosed. The syntenic groups were conserved in all the stocks. The relative abundance of repetitive DNA sequences was determined. C6, B11/L1Tc and E12 elements presented maximum 1.7-fold variation in copy number, whereas 195-bp satellite DNA (120,000 copies in Y strain) was four- to nine-fold more abundant in T. cruzi II stocks. The novel aspects of T. cruzi karyotype here presented contribute to the comprehension of the genome organization of this parasite and will assist the assignment of scaffold to the CL Brener chromosomal bands.

Animals↗
PubMed2004

Organizational variation of DYZ1 repeat sequences on the human Y chromosome and its diagnostic potentials.

The long arm of the human Y chromosome is flecked with various fractions of repetitive DNA. DYZ1 is one such fraction, which is organized tandemly as an array of a 3.4-kb repeat ranging from 2000-4000 copies in normal males. We have studied the organizational variation of the DYZ1 fraction on the human Y chromosome using DNA samples from CEPH family members and the random population employing the RFLP approach, fluorescence in situ hybridization (FISH), and conducted a similarity search with GenBank sequences. Typing of genomic DNA using DYZ1 as a probe showed an allele length and copy number variations even between two male siblings. Hybridization of DNA from monochromosome hybrids with this probe showed its presence on chromosome 15 in addition to the Y chromosome. Fluorescence in situ hybridization of metaphase chromosomes from an apparently normal male showed DYZ1 sequences in the proximal region of chromosome 11 in addition to the long arm of the Y chromosome. Typing of sets of semen and blood DNA samples from the same human individuals showed discernible allelic variation between the two samples, indicating tissue-specific programmed sequence modulation. DYZ1 seems to be the first probe having the unique potential to discriminate unequivocally the difference between the DNA originating from semen and blood samples, and may be exploited in forensic cases. This probe may also be used as a diagnostic tool to ascertain Y chromosome mosaicism in patients (e.g., Turner), its aberrant status in somatic cells, and possible sequence modulation/rearrangement in the germline samples. Additionally, this can be used to uncover sequence polymorphism in the human population.

Alleles↗
PubMed1992

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)

Analog-Digital Conversion↗
PubMed1982

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.

Animals↗
PubMed1990

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus).

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.

Animals↗
PubMed2005

Array comparative genomic hybridization and its applications in cancer.

Alteration in DNA copy number is one of the many ways in which gene expression and function may be modified. Some variations are found among normal individuals, others occur in the course of normal processes in some species and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur before or shortly after fertilization, and DNA dosage-alteration changes occurring in somatic cells are frequent contributors to cancer. Detecting these aberrations and interpreting them in the context of broader knowledge facilitates the identification of crucial genes and pathways involved in biological processes and disease. Over the past several years, array comparative genomic hybridization has proven its value for analyzing DNA copy-number variations. Here, we discuss the state of the art of array comparative genomic hybridization and its applications in cancer, emphasizing general concepts rather than specific results.

Gene Dosage↗
PubMed1988

Variation in copy number of a 24-base pair tandem repeat in the chloroplast DNA of Oenothera hookeri strain Johansen.

A highly variable region of chloroplast DNA has been analyzed from three isolates of Oenothera hookeri strain Johansen. The variability results from the presence of two, four or seven copies of a discrete 24-base pair tandem repeat in a segment of the chloroplast DNA within the inverted repeat. Alignment of this DNA region with the published tobacco cpDNA sequence shows that in Oenothera, the repeats are insertions within a large unidentified reading frame, with each repeat unit specifying an eight amino acid in-frame addition. A model to explain the frequent alterations in the copy number of this 24-bp unit is proposed: imprecise alignment and recombination between the two large inverted repeats followed by copy correction could result in an amplification or deletion of the 24-bp segments.

Base Sequence↗
PubMed2025

Absolute copy number aware CNV calling of sub-megabase segments in ultra-low coverage single-cell DNA sequencing data.

Recent advances in ultra-low coverage whole-genome sequencing (WGS) of single cells have enabled detailed analysis of copy number variation at a throughput approaching that of single-cell RNA sequencing. However, downstream computational methods have not seen comparable advances and are largely adaptations of deep sequencing methodology with reduced precision. Here, we present ASCENT, a computational method built to take full advantage of modern direct tagmentation-based WGS at ultra-low depth. Using joint segmentation with high-resolution bins, we accurately detect small segments, achieving accurate copy number profiles even at 100 000 reads per cell. ASCENT implements true absolute copy state inference for single cells, based on statistical modeling of coverage rather than comparison to a reference, while taking variable segment copy state into account. Further, ASCENT implements per-segment copy-neutral loss of heterozygosity (LOH) calling without the need for non-tumor or bulk WGS reference. When applied to a pediatric B-ALL sample, ASCENT finds copy-neutral LOH in a small segment and a minor subclone defined by breakpoints missed in bulk WGS. Thus, by applying appropriate computational methods, single-cell WGS provides clear advantages over bulk, even at a relatively low cell number and sequencing depth.

DNA Copy Number Variations↗