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Results for “Copy Number Variations”

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PubMed1998

Neurofibromatosis pseudogene amplification underlies euchromatic cytogenetic duplications and triplications of proximal 15q.

Cytogenetically visible interstitial duplications of proximal 15q, which lack the Prader-Willi Angelman critical region (PWACR) frequently segregate in families without phenotypic effect, but the nature of the extra euchromatin has remained unclear. We used comparative genome hybridisation to confirm that the extra material in a cytogenetic triplication originated from proximal 15q. A PAC clone containing sequences specific for the type-1 neurofibromatosis (NF-1) pseudogenes, which map to 15q11.2, hybridised along the length of the enlarged region between the PWACR and the centromere. Computerised measurement of the fluorescent signal from the enlarged and normal chromosomes gave an average ratio of 9.85:1, consistent with amplification. In a second family, an amplified P1-4 signal co-segregated with a cytogenetic duplication and the average ratio between amplified and normal signals in the proband was 8.22:1. Ratios in noncarrier family members and control individuals were close to unity in most cases, but significantly greater than one in at least one instance. Our results provide a novel explanation for cytogenetic variation in 15q11.2. They also suggest that NF-1 pseudogene copy number may be polymorphic in the normal population, and that high copy numbers can produce G bands which do not reflect those of the normal constitutional karyotype.

Adolescent↗

PubMed2004

Genomic dynamics of a low-copy-number satellite DNA family in Leptestheria dahalacensis (Crustacea, Branchiopoda, Conchostraca).

The LEP150 satellite DNA (satDNA) family found in Leptestheria dahalacensis (Ruppel, 1837) (Conchostraca) is a low-copy-number satellite with a canonical monomer of 150 bp. Nucleotide variation analyses suggest a 14-bp palindromic region as a possible protein binding site with constraints acting on the whole sequence but a 25-bp variable box. Besides the head-to-tail arrangement of 150 bp monomers, multimers analyses evidenced incomplete monomers, one duplication event, and three inversions. Both observed rearrangements and the higher values of sequence variability scored suggest that rearranged monomers reside in regions with a lower degree of homogenisation efficiency. Sixty-seven percent of the breakpoints occurs at kinkable dinucleotides, thus supporting their role in rearrangements as documented in alphoid satDNA recombination events. Monomers of different lengths may result from crossing over between repeats misaligned through the direct and inverted subrepeats of LEP150 monomers. ANOVA results indicate that the same range of sequence diversity is experienced at the individual and population ranks; therefore, the evolution of the L. dahalacensis satDNA is concerted.

Animals↗

PubMed2001

Quantitation of HCV RNA using real-time PCR and fluorimetry.

Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5' HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5' NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.

Fluorometry↗

PubMed1990

Identification of toxigenic Clostridium difficile strains by using a toxin A gene-specific probe.

A 4.5-kilobase PstI fragment encoding part of the toxin A gene was isolated and used as a DNA probe in colony hybridization studies with 58 toxigenic and 17 nontoxigenic Clostridium difficile strains. All 58 toxigenic strains showed positive hybridization, in contrast to the 17 nontoxigenic strains. Southern blot analysis with the toxin A gene probe showed hybridization to a single fragment of equal intensities for HindIII-digested genomic DNAs isolated from C. difficile strains of wide-ranging toxin production. The positive hybridization signals were due to fragments of heterogeneous lengths (9 to 13 kilobases) for toxigenic strains of different types but were absent for the nontoxigenic strains. These results suggest the presence of a single copy of the toxin A gene on the genome of C. difficile strains, and the wide variation of toxin expression is not a reflection of gene copy number. The lack of toxin activity for nontoxigenic strains can be explained by the absence of at least part of the toxin A gene. The toxin A gene probe was tested against clostridial strains from 18 other species, of which only toxigenic C. sordellii strains showed positive hybridization. The specificity of the toxin A gene probe for toxigenic strains may lead to improved methods for the specific identification of toxigenic C. difficile strains from clinical specimens.

Bacterial Toxins↗

PubMed1997

Performance of the Amplicor human immunodeficiency virus type 1 PCR and analysis of specimens with false-negative results.

Over a 4-year period, the Roche Amplicor kit was used in a United Kingdom reference laboratory for the detection or confirmation of human immunodeficiency virus (HIV) type 1 infection, particularly in infants born to HIV-infected mothers. Of 408 specimens from adults and older children tested, the 122 seronegative specimens were all Amplicor negative. Of the 286 seropositive specimens, 268 were Amplicor positive. On the basis of these results, the Amplicor assay has a specificity of 100% and a sensitivity of 93.7%. In addition, for 247 specimens from infants and young children, serological results may not have been diagnostic because of placental transfer of maternal antibodies. Forty-eight were Amplicor positive, and of the 199 Amplicor-negative specimens, 19 were assumed to be false negative on the basis of clinical data, serological markers (including p24 antigen), and/or results for previous or follow-up specimens. This represents a sensitivity of 75% for the Amplicor test for specimens from patients under 2 years of age. Of these 37 false-negative specimens plus 2 specimens from other laboratories, 31 could be characterized by amplifying extracted material from them by an in-house nested gag PCR spanning the Amplicor target region. The amplicons were sequenced and found to represent subtypes A (35.5%), B (22.6%), C (22.6%), D (16.1%), and G (3.2%). False-negative results by the Amplicor assay may be ascribed to low-target copy number, the physical behavior of one primer (SK462), and sequence variation in the target region of the other primer (SK431).

Adult↗

PubMed2006

Diagnosis and monitoring of chronic myeloid leukemia by qualitative and quantitative RT-PCR.

Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) methods for the quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia (CML) has become the predominant molecular monitoring technique. The BCR-ABL fusion gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, high-throughput method to accurately assess the level of treatment response and provides an early indication of emerging drug resistance. The ABI Prism 7700 Sequence Detection System uses TaqMan fluorogenic probes to quantitate specific nucleic acid sequences using RQ-PCR. The analyzer monitors an increase in fluorescence during the PCR cycle, which is proportional to the amount of accumulated product. The starting copy number is calculated relative to a series of standards. The copy number is normalized to a control gene that compensates for variations in the efficiency of the RT step and for the degree of RNA degradation. In our experience, reliable and consistent RQ-PCR requires thorough validation of all aspects of the procedure, including the selection of an appropriate control gene, careful assay design to avoid polymorphisms in primer or probe binding sites and to exclude the amplification of contaminating DNA, and monitoring the performance of the RQ-PCR by the use of quality control samples.

Cloning, Molecular↗

PubMed1993

Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR.

The PCR has proven to be useful in the analysis of gene expression of specific mRNAs. Although PCR is able to detect rare mRNA transcripts following reverse transcription PCR, determining relative or absolute copy number can be difficult due to sample-to-sample variation. The use of a recombinant mRNA internal standard that contains target mRNA primer sequences greatly improves reproducibility of quantitation. Reverse transcription PCR products generated from the internal standard can be distinguished from the product generated from the target gene mRNA because of their size difference. In this report we present a facile and general PCR-based method for synthesis of internal standards that may be used as competitive or co-amplified templates for quantitative reverse transcription PCR.

Animals↗

PubMed1997

Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups.

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.

Adult↗

PubMed2026

A De Novo 16p13.3 Triplication Underlying Early-Onset Complex Neurodegeneration.

BACKGROUND: Neurodegenerative disorders are clinically and genetically heterogeneous, characterized by progressive neuronal loss and multidomain functional decline. Despite a presumed genetic etiology, a substantial proportion of cases remain molecularly undiagnosed. OBJECTIVE: The aim was to identify the genetic cause of an early-onset neurodegenerative disorder presenting with ataxia and cognitive impairment. METHODS: Rare copy-number variants were detected via short-read whole-genome sequencing (WGS), with candidate structural models inferred using long-read WGS. We performed transcriptomic profiling of peripheral blood leukocytes by RNA sequencing, with validation using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: We identified a de novo copy-number gain at 16p13.3. Combined copy-number profiling and long-read WGS suggested a candidate model comprising a triplicated segment in tandem with a proximal duplication, joined to a distal duplication via an inverted junction. Transcriptomic analysis demonstrated significant upregulation of ATP6V0C, AMDHD2, and PDPK1. CONCLUSIONS: These findings support a role for structural variation in early-onset neurodegeneration and highlight the value of combining short-read copy-number profiling with long-read WGS to detect and characterize complex genomic rearrangements. © 2026 International Parkinson and Movement Disorder Society.

16p13.3↗

PubMed2026

scSNViz: visualization and analysis of cell-specific expressed SNVs.

MOTIVATION: Accurately characterizing expressed genetic variation at the single-cell level is essential for understanding transcriptional heterogeneity, allelic regulation, and mutational dynamics within complex tissues. However, few tools enable comprehensive visualization and quantitative analysis of expressed variants across individual cells. RESULTS: scSNViz is an R package for the exploration, quantification, and visualization of expressed single-nucleotide variants (SNVs) from cell-barcoded single-cell RNA sequencing (scRNA-seq) data. The software supports estimation of variant allele fractions, clustering of SNV expression profiles, and 2D and 3D visualization of individual SNVs or user-defined SNV groups. Beyond visualization, scSNViz facilitates investigation of cell-, cluster-, or lineage-specific variant expression patterns, as well as allelic dynamics including imprinting, random allele inactivation, and transcriptional bursting. It interoperates seamlessly with established single-cell frameworks-Seurat for clustering, Slingshot for trajectory inference, scType for cell-type annotation, and CopyKat for copy-number profiling-enabling integrative multi-omic analyses of expressed variation. AVAILABILITY AND IMPLEMENTATION: scSNViz is implemented in R and freely available at https://github.com/HorvathLab/scSNViz (DOI: 10.5281/zenodo.17307516). The package includes comprehensive documentation and example workflows designed for users with limited bioinformatics experience.

Software↗

PubMed1997

Estimating the age of alleles by use of intraallelic variability.

A method is presented for estimating the age of an allele by use of its frequency and the extent of variation among different copies. The method uses the joint distribution of the number of copies in a population sample and the coalescence times of the intraallelic gene genealogy conditioned on the number of copies. The linear birth-death process is used to approximate the dynamics of a rare allele in a finite population. A maximum-likelihood estimate of the age of the allele is obtained by Monte Carlo integration over the coalescence times. The method is applied to two alleles at the cystic fibrosis (CFTR) locus, deltaF508 and G542X, for which intraallelic variability at three intronic microsatellite loci has been examined. Our results indicate that G542X is somewhat older than deltaF508. Although absolute estimates depend on the mutation rates at the microsatellite loci, our results support the hypothesis that deltaF508 arose < 500 generations (approximately 10,000 years) ago.

Alleles↗

PubMed2001

Population analysis of an unusual NOR-site polymorphism in brown trout (Salmo trutta L.).

A population analysis of an unusual NOR-site polymorphism previously detected in brown trout (Salmo trutta L.) from North-western Spain was carried out in 225 individuals from 20 native populations from this area. The analysis performed has permitted us to reveal: (i) the ubiquity of this phenomenon in most river basins from NW Spain, 13 extra-NORs not observed in standard trout being detected; (ii) the rDNA constitution of all extra-NORs, as confirmed by CMA3-staining and rDNA-FISH, and their capability to constitute their own nucleolus; (iii) the constant location of extra-NORs within individuals and their stable transmission across generations; and (iv) the telomeric location of Ag-NORs, which were randomly distributed in the karyotype of S. trutta, mostly in the heterozygous condition. Repetitive sequences in the IGS or scattered along rDNA units, rather than chromosome rearrangements, could play an important role in the dispersion of NORs. The polymorphism described seems to have a single geographical origin, since a positive correlation was demonstrated between the degree of polymorphism and geographical distance to a central point of the river basin where the phenomenon showed a higher intensity. The distribution of NOR-site variation as compared with allozyme variation in the populations studied, showed significant statistical differences. Selection against high copy number, or mutation due to changes in NOR location, could be acting on this polymorphism to explain the observed distribution. The high dispersion of NOR-site variants within and among populations suggests the transposition phenomenon responsible for NOR jumping could be still active.

Animals↗

PubMed1996

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Actins↗

PubMed2004

[Detection of HPV16 E6 gene in cervical tissues by quantitative polymerase chain reaction].

OBJECTIVE: To establish a method for detection of Human Papillomavirus (HPV) type16 E6 gene in Cervical carcinomas Specimens. To study the relationship between the quantities of HPV16 E6 (Human papillomavirus type16 E6 gene) in cervical tissues and the course of cervical disease in Xinjiang. METHODS: HPV16E6 gene and beta-actin was detected in parallel by FQ-PCR (fluorescence quantitative PCR). The number of copies of HPV16 E6 gene and beta-actin was detected in parallel by FQ-PCR (fluorescence quantitative PCR) in tissues of 69 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis and samples of 96 cervical smear samples of vaginitis and cervicitis. The variation in HPV copies per genomic DNA equivalent can be estimated by dividing the HPV copy number by the beta-actin copy number. RESULTS: The positive rate of HPV16 E6 gene was 83.0%, 75.7%, 93.3% and 3.3% in tissues of cervical cancer, cervical intraepithelial neoplasia (CIN), chronic cervicitis and samples of cervical smear respectively. The amount of HPV16 E6 gene was gradually higher by the developing of the course of cervical disease. They have positive rank correlation, r = 0.83, P < 0.01. CONCLUSION: The study underscores the importance of the relationship between the HPV16 E6 gene and the course of cervical disease in Xinjiang. It also suggests that the quantification of HPV16 E6 gene may be useful as a prognostic tool to identify women who are at increased risk of developing cervical cancer. This method may be applied to studies of a number of issues related to the natural history of cervical cancer, such as the amounts of HPV in high- and low-grade lesions.

Cervix Uteri↗

PubMed2001

Echinococcus granulosus: intraspecific genetic variation assessed by a DNA repetitive element.

A 186 bp Echinococcus granulosus-specific repetitive element, TREg, was used to assess genetic variation between strains. In G7 genotype (pig strain) it has the characteristics of a satellite DNA element with a copy number of 23000 per haploid genome. Analysis, by sequencing of TREg monomers, showed a great degree of identity within them. In the G1 genotype (common sheep strain) TREg-like repetitive elements were found in an interspersed distribution throughout the genome and in only 120 copies. The sequences of these monomers showed a great degree of variation between them and with TREg of G7 origin. The G6 genotype (camel strain) showed a pattern of distribution and copy number similar to the G7 genotype, and the G2 genotype (Tasmanian sheep strain) similar to the G1 genotype. Isolates from the G5 (cattle strain) and G4 (horse strain) genotypes also showed unique hybridization patterns in Southern blot experiments. The genomic plasticity of E. granulosus, which may have important consequences in the epidemiology and control of cystic hydatid disease is reflected in the results of this work.

Animals↗

PubMed2006

Contour area filtering of two-dimensional electrophoresis images.

We describe an algorithm, Contour Area Filtering, for separating background from foreground in gray scale images. The algorithm is based on the area contained within gray scale contour lines. It can be viewed as a form of local thresholding, or as a seed growing algorithm, or as a type of watershed segmentation. The most important feature of the algorithm is that it uses object area to determine the segmentation. Thus, it is relatively impervious to brightness and contrast variations across an image or between different images. Contour Area Filtering was designed specifically for image analysis of 2D electrophoresis gels, although it can be applied to other gray scale images. A typical gel image is an electrophoretogram or a phosphor image of 1000-2500 spots representing protein or DNA restriction fragments. The images are quantitative with spot intensities reflective of the number of proteins or the DNA fragment copy number. The background intensity can vary widely across the image caused both by variation in spot density and by the physical laboratory process of creating a gel. Analyzing and comparing gel images entails extracting and segmenting spots, registering images and matching spots, and measuring differences between spots. We present experimental results which show that Contour Area Filtering is a quick, efficient method for separating electrophoresis gel background from foreground with extremely high accuracy.

Algorithms↗

PubMed1996

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

Amino Acid Oxidoreductases↗

PubMed1994

Relationship between loss of estrogen and progesterone receptor expression and of 6q and 11q chromosome arms in breast cancer.

The expression of estrogen (ER) and progesterone (PR) receptors was quantified in a series of 95 cytogenetically characterized breast cancers. The relationship between deficiencies of 6q and 11q arms (where genes for ER and PR are mapped, respectively) and ER and PR expression was analyzed. The range of variations in expression was very large by comparison to that of the number of chromosome arms. Furthermore, low expression without chromosome loss and high expression with chromosome loss were occasionally observed. Thus, major variations in ER and PR expression were independent of the number of copies of the corresponding gene. However, both the decrease in ER expression and 6q arm losses were correlated with cytogenetic evolution of the tumors, this correlation being less significant for PR and the 11q arm. In addition, 6q- but not 11q- tumors have, on the average, a low ER/PR ratio, whereas 11q- but not 6q- tumors have a low PR/ER ratio. When all 6q- or 11q- tumors were compared to those with no 6q or 11q losses, the average ER or PR value of the former was about half of the later. These data suggest that, in addition to a regulatory change of non-genetic origin, gene dosage effect plays a secondary, but significant, additional role.

Breast Neoplasms↗