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Results for “Copy Number Variations”

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PubMed1988

Unequal partitioning of bovine mitochondrial genotypes among siblings.

Two polymorphic mitochondrial DNA genomes, differing by a single Hpa II restriction site, are present at significantly different levels in tissue of three sibling dairy cows. The relative ratio of the two heteroplasmic molecules varies 3-fold among these three animals and documents a rapid segregation of mitochondrial genotypes in mammals. DNA sequencing shows the difference is due to a single guanine at position 364 in bovine mitochondrial DNA. A model involving unequal partitioning of the two amplified mitochondrial DNA species during the early cell divisions of the embryo can explain the appearance of such variation in heteroplasmic sibling animals. The model provides a basis for understanding the rapid DNA sequence variation observed in vertebrate mitochondrial DNA despite its high copy number and strict maternal inheritance.

Animals↗

PubMed1993

Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae.

The chromatin structure of yeast ribosomal DNA was analyzed in vivo by crosslinking intact cells with psoralen. We found that in exponentially growing cultures the regions coding for the 35S rRNA precursor fall into two distinct classes. One class was highly accessible to psoralen and associated with nascent RNAs, characteristic for transcriptionally active rRNA genes devoid of nucleosomes, whereas the other class showed a crosslinking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rRNA gene copies. By crosslinking the same strain growing in complex or minimal medium, we have shown that yeast cells can modulate the proportion of active (non-nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions which suggests that yeast can regulate rRNA synthesis by varying the number of active gene copies, in contrast to the vertebrate cells studied so far. Whereas intergenic spacers flanking inactive rRNA gene copies are packaged in a regular nucleosomal array, spacers flanking active genes show an unusual crosslinking pattern suggesting a complex interaction of regulatory factors and histones with DNA.

Chromatin↗

PubMed2025

MarkerMatch: A Proximity-Based Probe-Matching Algorithm for Joint Analysis of Copy-Number Variants from Different Genotyping Arrays.

MOTIVATION: Copy-number variants (CNVs) are a form of genetic structural variation with increasing importance in complex human disorders. Both DNA sequencing and microarray data can be used to call CNVs, which can be used in association tests, such as association between CNV number and disease status. Unlike genotypes, CNV detection in microarrays requires the use of observed intensity signals at each probe, which limits the imputability for analyses that span multiple array types. Thus far, a consensus set of probes (the intersection encompassing the probes that occur in common on all arrays) has been used to circumvent the problem of differing array-specific sensitivities. This has, however, led to excessive reduction in overall sensitivity of CNV calls as arrays can have an undesirably low overlap of probe sets. To overcome this limitation, we developed MarkerMatch, a proximity-based algorithm that matches probes across different genotyping microarrays to maximize the number of probes considered in the CNV calling algorithm, thereby increasing the resolution and sensitivity while preserving precision. RESULTS: By analyzing CNV calls from 4,906 individuals genotyped across three different arrays (Global Screening Array, Omni2.5 array, and Omni Express Exome array), we show that the MarkerMatch approach improves sensitivity by increasing the density of probes available for CNV calling while maintaining precision or improving it relative to the current practice (e.g., use of consensus probes only). We further demonstrate that MarkerMatch exceeds the output from current practice in terms of F1 score, Fowlkes-Mallows index, and Jaccard index. We also optimize MarkerMatch parameters, D MAX and Method, and find an optimal D MAX setting at 10kb, with no clear optimal candidate based on Method, indicating that parameters for this metric should be determined on a use case basis.

Journal Article↗

PubMed2006

Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry in genomics research.

The beginning of this millennium has seen dramatic advances in genomic research. Milestones such as the complete sequencing of the human genome and of many other species were achieved and complemented by the systematic discovery of variation at the single nucleotide (SNP) and whole segment (copy number polymorphism) level. Currently most genomics research efforts are concentrated on the production of whole genome functional annotations, as well as on mapping the epigenome by identifying the methylation status of CpGs, mainly in CpG islands, in different tissues. These recent advances have a major impact on the way genetic research is conducted and have accelerated the discovery of genetic factors contributing to disease. Technology was the critical driving force behind genomics projects: both the combination of Sanger sequencing with high-throughput capillary electrophoresis and the rapid advances in microarray technologies were keys to success. MALDI-TOF MS-based genome analysis represents a relative newcomer in this field. Can it establish itself as a long-term contributor to genetics research, or is it only suitable for niche areas and for laboratories with a passion for mass spectrometry? In this review, we will highlight the potential of MALDI-TOF MS-based tools for resequencing and for epigenetics research applications, as well as for classical complex genetic studies, allele quantification, and quantitative gene expression analysis. We will also identify the current limitations of this approach and attempt to place it in the context of other genome analysis technologies.

Animals↗

PubMed2001

Viral load test reports: a description of content from a sample of US laboratories.

CONTEXT: Human immunodeficiency virus (HIV) RNA testing (viral load testing) is increasingly important in the care of patients infected with HIV-1 to determine when to initiate, monitor, and change antiretroviral therapy. Patient viral load testing information is communicated to the clinician through the laboratory test report. OBJECTIVES: To examine the format and information used in reporting viral load testing results and determine the clarity of the information provided in these reports. DESIGN: Patient test reports with all personal identifiers removed were requested of viral load testing laboratories participating in a telephone survey of laboratory practices. Hospital, independent, health department, and "other type" laboratories identified as university-associated laboratories participated in the telephone survey. RESULTS: Thirty-seven unique test reports were collected. All laboratories reported results in copies/mL, while 14% also reported results as "log(10) copies/mL." The test kit was identified by only 24% of the laboratories. Reportable ranges were specified by 70% of the laboratories, but there was considerable variation in terminology. One laboratory reported a viral load copy number below the manufacturer's test kit lower limit of sensitivity. The layout and format differed among reports. Some results were expressed in log(10), others contained nonsignificant integers, while others contained exponential numbers. Supplemental information in some reports included previous patient test results and significance of changes from baseline. The format of some reports made it difficult to read the report information and interpret the testing results. CONCLUSION: This study emphasizes the importance of standardizing the reporting of HIV-1 viral load test results to minimize result misinterpretation and incorrect treatment.

Data Collection↗

PubMed1990

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number.

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.

Chloramphenicol O-Acetyltransferase↗

PubMed1999

A theoretical study of random segregation of minicircles in trypanosomatids.

The kinetoplast (k) DNA network of trypanosomatids is made up of approximately 50 maxicircles and the order of 10(4) minicircles. It has been proposed, based on various observations and experiments, that the minicircles are randomly segregated between daughter cells when the parent cell divides. In this paper, this random segregation hypothesis is theoretically tested in a population dynamics model to see if it can account for the observed phenomena. The hypothesis is shown to successfully explain, in Leishmania tarentolae, the observation that there are a few major and many minor minicircle classes, the fluctuations of minicircle class copy numbers over time, the loss of non-essential minicircle classes, the long survival times of a few of these classes and that these classes are likely to be the major classes within the population. Implications of the model are examined for trypanosomatids in general, leading to several predictions. The model predicts variation in network size within a population, variation in the average network size and large-scale changes in class copy number over long time-scales, an evolutionary pressure towards larger network sizes, the selective advantage of non-random over random segregation, very strong selection for the amplified class in Crithidia fasciculata if its minicircles undergo random segregation and that Trypanosoma brucei may use sexual reproduction to maintain its viability.

Animals↗

PubMed1993

Scaffold attachment regions increase reporter gene expression in stably transformed plant cells.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Base Sequence↗

PubMed1992

Fitness effects of Ty transposition in Saccharomyces cerevisiae.

It has been suggested that the primary evolutionary role of transposable elements is negative and parasitic. Alternatively, the target specificity and gene regulatory capabilities of many transposable elements raise the possibility that transposable element-induced mutations are more likely to be adaptively favorable than other types of mutations. Populations of Saccharomyces cerevisiae containing large amounts of variation for Ty1 genomic insertions were constructed, and the effects of Ty1 copy number on two components of fitness, yield and growth rate were determined. Although mean stationary phase density decreased with increased Ty1 copy number, the variance and range increased. The distributions of stationary phase densities indicate that many Ty1 insertions have negative effects on fitness, but also that some may have positive effects. To test directly for adaptively favorable Ty1 insertions, populations containing large amounts of variability for Ty1 copy number were grown in continuous culture. After 98-112 generations the frequency of clones containing zero Ty1 elements had decreased to approximately 0.0, and specific Ty1-containing clone families had predominated. Considering that most of the genetic variation in the populations was due to Ty1 transposition, and that Ty1 insertions had, on average, a negative effect on fitness, we conclude that Ty1 transposition events were directly responsible for the production of adaptive mutations in the clones that predominated in the populations.

Blotting, Southern↗

PubMed1993

Regulation of the expression of mitochondrial proteins: relationship between mtDNA copy number and cytochrome-c oxidase activity in human cells and tissues.

The relationship between the relative amounts of nuclear and mitochondrial genes for cytochrome-c oxidase subunits and their transcripts and cytochrome-c oxidase activity was investigated in several human tissues and cell lines to get more insight into the regulation of the expression of this mitochondrial enzyme complex. The results show: (1) a wide range of mtDNA copy numbers; (2) constant ratios between the steady-state levels of the transcripts for the various cytochrome-c oxidase subunits, and (3) large variations in cytochrome-c oxidase activity in different tissues and cell lines that could not be related to the differences in mtDNA copy number. We conclude that the transcription of genes for both mitochondrial and nuclear cytochrome-c oxidase subunits is regulated coordinatedly, but also that the mtDNA copy number plays a minor role in determining differences in cytochrome-c oxidase activity between different cell and tissue types.

Cell Nucleus↗

PubMed2006

Genome assembly comparison identifies structural variants in the human genome.

Numerous types of DNA variation exist, ranging from SNPs to larger structural alterations such as copy number variants (CNVs) and inversions. Alignment of DNA sequence from different sources has been used to identify SNPs and intermediate-sized variants (ISVs). However, only a small proportion of total heterogeneity is characterized, and little is known of the characteristics of most smaller-sized (<50 kb) variants. Here we show that genome assembly comparison is a robust approach for identification of all classes of genetic variation. Through comparison of two human assemblies (Celera's R27c compilation and the Build 35 reference sequence), we identified megabases of sequence (in the form of 13,534 putative non-SNP events) that were absent, inverted or polymorphic in one assembly. Database comparison and laboratory experimentation further demonstrated overlap or validation for 240 variable regions and confirmed >1.5 million SNPs. Some differences were simple insertions and deletions, but in regions containing CNVs, segmental duplication and repetitive DNA, they were more complex. Our results uncover substantial undescribed variation in humans, highlighting the need for comprehensive annotation strategies to fully interpret genome scanning and personalized sequencing projects.

Base Sequence↗

PubMed1988

The cDNA of cucumber mosaic virus-associated satellite RNA has in vivo biological properties.

Two isolates of cucumber mosaic virus (CMV)-associated satellite RNA, differing in their biological properties, have been reverse transcribed. One was able to induce the tomato necrotic syndrome whereas the other one attenuated fernleaf symptoms on tomato plants after co-inoculation with the helper virus. cDNAs representing partial or full-length copies have been cloned in the plasmid pAT 153 and sequenced. The two RNAs showed a very limited number of variations (2 to 5 substitutions depending on the clones and a one base deletion). Full-length cDNA copies possessed the same biological properties that characterized the parent satellite RNA. Efficiency of the cDNA depended upon its form in the inoculum (circular or linear plasmid or excised cDNA) and upon the form of the helper virus (viral RNAs or virions) with which it seemed to compete for installation and/or expression.

Base Sequence↗

PubMed2000

Patterns of ribosomal gene variation in elite commercial chicken pure line populations.

The nucleolus organizer region (NOR) encodes the tandemly repeated 18S, 5.8S and 28S ribosomal (r) RNA genes. The NORs of broiler and layer commercial chicken pure lines were studied to establish the type and extent of genetic variation at this important locus. The parameters studied were gene copy number, repeat size, and diversity of NOR-types. The populations were organized into three groups for analysis including brown-egg broiler (13 lines), brown-egg layer (six lines), and white-egg layer (eight lines). The ribosomal gene copy number average of the white-egg layer populations was significantly lower (329 genes) than that of the brown-egg layers (372 genes); the brown-egg broiler ribosomal gene average was intermediate (350 genes). The white-egg layer populations exhibited a ribosomal repeat unit average size of 36 kb, significantly different from the brown-egg layer and brown-egg broiler average repeat unit size of 32.5 and 33.9 kb, respectively. NOR array size was similar among the three groups (6 mb). The brown-egg broiler populations exhibited polymorphic NOR patterns, intra- and interline, whereas the white-egg layer populations were essentially monomorphic for NOR-type; brown-egg layers exhibited an intermediate level of NOR diversity. Some NOR array characteristics may be a function of breed origin as brown-egg commercial populations, both broilers and layers, have similar breed origins and exhibited similarities for predominant repeat unit size as compared with white-egg layer populations. However, the finding that brown-egg broiler lines typically exhibit a greater number of segregating NOR-types than brown-egg layer lines suggests that the selection schemes of broiler vs. layer pure line populations may also have influenced the degree of variation at this gene complex.

Animals↗

PubMed2004

Possible gene dosage effect of glutathione-S-transferases on atopic asthma: using real-time PCR for quantification of GSTM1 and GSTT1 gene copy numbers.

Asthma is a complex genetic disorder characterized by chronic inflammation in the airways. As oxidative stress is a key component of inflammation, variations in genes involved in antioxidant defense could therefore be likely candidates for asthma. Three enzymes from the superfamily glutathione-S-transferase (GST) involved in the antioxidant defense were tested for association to asthma using 246 Danish atopic families in a family-based transmission disequilibrium test (TDT) design. A real-time PCR assay for relative quantification of gene copy number of GSTM1 and GSTT1 was developed. The assay made it possible to distinguish individuals with zero, one, and two copies and thereby to investigate whether the GST genes influenced susceptibility to asthma in a dose-dependent manner. We found that asthmatic patients with two copies of GSTM1 were significantly underrepresented (p<0.0005) and the significance increased by 10-fold when only atopic asthmatics were analyzed (p<0.00005). GSTT1 was significantly associated in an additive model to asthma, in which the alleles carrying the deletion of the gene were transmitted to affected offspring more often than expected by chance (p=0.019). The same transmission disequilibrium of the null GSTT1 allele was seen in patients with atopic asthma (p=0.021). The polymorphism c.342A>G (p.I105V) in GSTP1 has previously been suggested as a risk factor for asthma. However, significant association with asthma or related atopic phenotypes could not be established in our study. We conclude that deletions of GSTM1 and GSTT1 could be risk factors for asthma and that the genes might have a protective role in the development of atopic asthma.

Alleles↗

PubMed1998

Evolution of multigene families by gene duplication. A haploid model.

Evolution of multigene families by gene duplication and subsequent diversification is analyzed assuming a haploid model without interchromosomal crossing over. Chromosomes with more different genes are assumed to have higher fitness. Advantageous and deleterious mutations and duplication/deletion also affect the evolution, as in previous studies. In addition, negative selection on the total number of genes (copy number selection) is incorporated in the model. First, a Markov chain approximation is used to obtain formulas for the average numbers of different alleles, genes without pseudogene mutations, and pseudogenes assuming that mutation rates and duplication/deletion rates are all very small. Computer simulation shows that the approximation works well if the products of population size with mutation and duplication/deletion rates are all small compared to 1. However, as they become large, the approximation underestimates gene numbers, especially the number of pseudogenes. Based on the approximation, the following was found: (1) Gene redundancy measured by the average number of redundant genes decreases as advantageous selection becomes stronger. (2) The number of different genes can be approximately described by a linear pure-birth process and thus has a coefficient of variation around 1. (3) The birth rate is an increasing function of population size without copy number selection, but not necessarily so otherwise. (4) Copy number selection drastically decreases the number of pseudogenes. Available data of mutation rates and duplication/deletion rates suggest much faster increases of gene numbers than those observed in the evolution of currently existing multigene families. Various explanations for this discrepancy are discussed based on our approximate analysis.

Alleles↗

PubMed2005

Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis.

In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

Gene Expression Profiling↗

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↗