SEARCH · PubMed Health

Results for “DNA Copy Number Variations”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources

PubMed2000

Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study.

Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.

Antimetabolites, Antineoplastic↗

PubMed1994

Evidence for Darwinian selection of the 2-micron plasmid STB locus in Saccharomyces cerevisiae.

The 2-microns plasmid of industrial and laboratory strains of Saccharomyces cerevisiae exists as two main polymorphic forms designated type I and type II. Polymorphism is restricted to the 3200-bp right unique region where types I and II show approximately 10% nucleotide divergence in trans-acting REP1 and RAF loci and 30% divergence in the cis-acting STB locus. In addition, the cis-acting STB plasmid partition locus of type II plasmids varies in sequence and copy number of a 125-bp repeat. We devised chimeric and 2-microns plasmid stability experiments to evaluate the effect of STB polymorphism on plasmid fitness in amphiploid industrial and haploid laboratory strains. Reciprocal experiments of type-II STB chimeric plasmids in type-I bakers' yeast or a type-I chimeric plasmid in type-II distillers', wine, or haploid strains showed similar partition efficiencies. However, chimeric and 2-microns plasmids carrying a 250-bp STB from a type-II haploid strain had reduced fitness in a type-II industrial wine strain. These results in conjunction with molecular analyses of 2-microns-like and 2-microns plasmids indicates the coevolution of STB with trans-acting plasmid and host-cell factors.

Biological Evolution↗

PubMed1999

Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver.

It is unclear whether the sequence populations of hepatitis C virus (HCV) quasispecies in the liver and in serum are different, as a variety of studies on this subject provide conflicting results. In the current study, the populations of HCV 5' untranslated region (5' UTR) sequences in paired serum and liver samples from six patients with chronic hepatitis were analysed. Liver-derived, negative-strand viral RNA was amplified with a highly strand-specific Tth-based assay, and extensive measures, including accounting for template copy number, were undertaken to lower the risk of sporadic artefactual polymorphism. Amplified sequences were compared by single-strand conformation polymorphism analysis and by direct sequencing of identified differences. In four patients, liver samples were found to contain variants within the quasispecies which were not found in serum or negative-strand viral RNA, while in the remaining two patients, low virus titre prevented a reliable quasispecies analysis. These results suggest the presence in the same individual of HCV variants differing in the 5' UTR and possibly replicating with different kinetics.

5' Untranslated Regions↗

PubMed2004

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization.

Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Tumor biopsies were collected from 112 patients diagnosed with early breast cancer from January 1990 to March 1994. The samples were analyzed for HER2 DNA amplification by real-time PCR on LightCycler and by FISH and for HER2 protein expression by IHC. Inter-assay variation for HER2 measured by LightCycler was 10% (x =3.1; n=17). Amplification > or = 2 was observed in 19% of the patients. Concordance rates between real-time PCR and the other methods were 91% (IHC) and 92% (FISH). The correlation between real-time PCR and FISH was highly significant (p < 0.001). The "LightCycler-HER2/neu DNA quantification kit" produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real-time PCR compares with FISH and IHC. The data show that results obtained for amplification of HER2 by real-time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH.

Awards and Prizes↗

PubMed1992

Predictions of a 2-locus model for disease heterogeneity: application to adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked disorder that exhibits a wide range of phenotypic variability within individuals in a single family carrying the mutant allele. A 2-locus epistatic model has been proposed to explain the inheritance of the severe childhood form of ALD and the milder adult-onset adrenomyloneuropathy (AMN). Under a dominant epistatic model, a single M allele at an autosomal modifier locus ameliorates the most severe effects of the disease allele leading to the milder AMN phenotype; only males with genotype mm would have ALD. Under a recessive epistatic model, 2 copies of the M allele would be necessary to have the milder AMN phenotype. Here, we show that recurrence risks for a second affected male depend on the frequency of the protective allele at this modifier locus. Whereas it is most likely that 2 affected brothers will be concordant for their disease phenotypes, discordant pairs of affected brothers are possible at all frequencies of M. Within a narrow range of modifier allele frequencies, the predicted distribution of affected sib pairs (over all families) is consistent with empiric data from a large clinic population. Here we suggest sampling discordant affected sib pairs as a strategy for detecting linkage between a polymorphic DNA marker and a possible modifier gene. Since both epistatic models predict that discordant affected pairs should not share 2 alleles at the modifier locus, we expect that departures from the null distribution could be detected with relatively small numbers of sib pairs.

Adrenoleukodystrophy↗

PubMed1992

A fourth family of the Plasmodium falciparum S-antigen.

The S-antigen of Plasmodium falciparum is a highly diverse heat stable protein that is located in the parasitophorous vacuole of the mature asexual intraerythrocytic parasite. The gene for S-antigen exists within the parasite population as multiple alleles at a single locus. Its sequence contains a large central block of tandemly arranged peptides that are identical or very similar in one allele but differ widely in sequence, repeat length and number among different alleles, and consequently antigenic specificity. Thus, antibodies directed against the repeat region can be used to define the serotype of an S-antigen. Flanking this repeat block are 2 short regions of non-repetitive sequence which have been described as occurring in three different forms, each of which is used to define a single S-antigen family. We present the S-antigen sequence for the isolate 3D7 which defines not only a novel serotype but also a novel S-antigen family. The central repeat block is composed of 57 copies of an 8-residue peptide with consensus sequence ED(E/K)VSNG(R/G). Comparison of the four S-antigen families reveals that they differ considerably from each other with variation being most pronounced in the carboxy terminal-flanking region. This pattern of sequence variation differs considerably from that found for MSA-1 and MSA-2, the only other diverse proteins of P. falciparum for which sequence information is available.

Amino Acid Sequence↗

PubMed2001

Positive selection of a gene family during the emergence of humans and African apes.

Gene duplication followed by adaptive evolution is one of the primary forces for the emergence of new gene function. Here we describe the recent proliferation, transposition and selection of a 20-kilobase (kb) duplicated segment throughout 15 Mb of the short arm of human chromosome 16. The dispersal of this segment was accompanied by considerable variation in chromosomal-map location and copy number among hominoid species. In humans, we identified a gene family (morpheus) within the duplicated segment. Comparison of putative protein-encoding exons revealed the most extreme case of positive selection among hominoids. The major episode of enhanced amino-acid replacement occurred after the separation of human and great-ape lineages from the orangutan. Positive selection continued to alter amino-acid composition after the divergence of human and chimpanzee lineages. The rapidity and bias for amino-acid-altering nucleotide changes suggest adaptive evolution of the morpheus gene family during the emergence of humans and African apes. Moreover, some genes emerge and evolve very rapidly, generating copies that bear little similarity to their ancestral precursors. Consequently, a small fraction of human genes may not possess discernible orthologues within the genomes of model organisms.

Animals↗

PubMed1987

KP elements repress P-induced hybrid dysgenesis in Drosophila melanogaster.

Molecular and genetic analysis has revealed a specific P factor deletion derivative (the KP element) which is able to repress P-induced hybrid dysgenesis. All naturally occurring strains lacking the P cytotype (M') that were examined, throughout the world contain up to 30 copies of KP per haploid genome together with complete P factors. The KP element is derived from the P factor by an internal deletion of 1753 bp removing nucleotides 808-2560 and is transcribed to yield an abundant 0.8-kb poly(A)+ RNA with the coding capacity for an in-frame 207 amino acid polypeptide. Genetic crosses show that KP elements preferentially accumulate in the presence of P factors and suppress hybrid dysgenesis. Suppression is transmitted through both sexes and is thus distinct from the maternally transmitted P cytotype mode of suppression. The spread of KP elements is probably due to the continual selection of individuals with the highest numbers of KP elements in which P-induced hybrid dysgenesis is suppressed.

Animals↗

PubMed1993

Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine.

Deficient hydroxylation of debrisoquine is an autosomal recessive trait that affects approximately 7% of the Caucasian population. These individuals (poor metabolizers) carry deficient CYP2D6 gene variants and have an impaired metabolism of severely commonly used drugs. The opposite phenomenon also exists, and certain individuals metabolize the drugs very rapidly, resulting in subtherapeutic plasma concentrations at normal doses. In the present study, we have investigated the molecular genetic basis for ultrarapid metabolism of debrisoquine. Restriction fragment length polymorphism analysis of the CYP2D locus in two families with very rapid metabolism of debrisoquine [metabolic ratio (MR) for debrisoquine = 0.01-0.1] revealed the variant CYP2D6 gene CYP2D6L. Eco RI RFLP and Xba I pulsed-field gel electrophoresis analyses showed that this gene had been amplified 12-fold in three members (father and his two children) of one of the families, and two copies were present among members of the other family. The CYP2D6L gene had an open reading frame and carried two mutations causing amino acid substitutions: one in exon 6, yielding an Arg-296-->Cys exchange and one in exon 9 causing Ser-486-->Thr. The MR of subjects carrying one copy of the CYP2D6L gene did not significantly differ from that of those with the wild-type gene, indicating that the structural alterations were not of importance of the catalytic properties of the gene product. Examination of the MR among subjects carrying wild-type CYP2D6, CYP2D6L, or deficient alleles revealed a relationship between the number of active genes and MR. The data show the principle of inherited amplification of an active gene. Furthermore, the finding of a specific haplotype with two or more active CYP2D6 genes allows genotyping for ultrarapid drug metabolizers. This genotyping could be of predictive value for individualized and more efficient drug therapy.

Alleles↗

PubMed1999

Transposable elements and genome evolution: the case of Drosophila simulans.

Drosophila simulans presents a large variation in copy number among various transposable elements (TEs) and among natural populations for a given element. Some elements such as HMS beagle, blood, flea, tirant, coral, prygun, jockey, F, nomade and mariner are absent in most populations, except in one or two which have copies on their chromosome arms. This suggests that some TEs are being awakened in D. simulans and are in the process of invading the species while it is colonizing the world. The elements 412 and roo/B104 present a wide insertion polymorphism among D. simulans populations, but only the 412 copy number follows a temperature cline. One population (Canberra from Australia) has a very high copy number for the 412 element and for many other TEs as well, indicating that some populations may have lost control of some of their TEs. While the 412 transposition rate is similar in all populations, its transcription level throughout developmental stages varies with populations, depending on copy number. Populations with 412 copy number higher than 10-12 exhibit co-suppression, while the expression in populations with lower numbers depends on the insertion location. All these results suggest genomic invasions by 412 and other TEs during the worldwide spread of the D. simulans species.

Animals↗

PubMed1998

Multiple recombination events maintain sequence identity among members of the nitrogenase multigene family in Rhizobium etli.

A distinctive characteristic of the Rhizobium genome is the frequent finding of reiterated sequences, which often constitute multigene families. Interestingly, these families usually maintain a high degree of nucleotide sequence identity. It is commonly assumed that apparent gene conversion between reiterated elements might lead to concerted variation among members of a multigene family. However, the operation of this mechanism has not yet been demonstrated in the Rhizobiaceae. In this work, we employed different genetic constructions to address the role of apparent gene conversion as a homogenizing mechanism between members of the plasmid-located nitrogenase multigene family in Rhizobium etli. Our results show that a 28-bp insertion into one of the nitrogenase reiterations can be corrected by multiple recombination events, including apparent gene conversion. The correction process was dependent on the presence of both a wild-type recA gene and wild-type copies of the nitrogenase reiterations. Frequencies of apparent gene conversion to the wild-type nitrogenase reiterations were the same when the insertion to be corrected was located either in cis or in trans, indicating that this event frequently occurs through intermolecular interactions. Interestingly, a high frequency of multiple crossovers was observed, suggesting that these large plasmid molecules are engaging repeatedly in recombination events, in a situation akin to phage recombination or recombination among small, high-copy number plasmids.

Base Sequence↗

PubMed2005

Inferences of evolutionary relationships from a population survey of LTR-retrotransposons and telomeric-associated sequences in the Saccharomyces sensu stricto complex.

The Saccharomyces sensu stricto complex consists of six closely related species and one natural hybrid. Intra- and inter- species variability in repetitive elements can help elucidate the population structure and evolution of these close relatives. The chromosome positions of several telomeric associated sequences (TASs) and LTR-retrotransposons have been determined, using PFGE, in 112 isolates. Most of the repetitive elements studied are found in multiple copies in each strain, although in some subpopulations these elements are present in low copy number or are absent. Hybridization patterns and copy numbers of the repetitive elements correlate with geographic distribution. These patterns may yield interesting clues as to the origins and evolution of some TASs and retrotransposons, e.g. we can infer that Y' originated on the left end of chromosome XIV. There is strong evidence for horizontal transfer of Ty2 between S. cerevisiae and S. mikatae. Ty1 and Ty5 are either lost easily or frequently horizontally transferred. We have also found some gross chromosomal rearrangements in isolates within species and a few new natural hybrids between species, indicating that these processes occur in the wild and are not limited to conditions of human influence. DNA sequences have been deposited with the EMBL/GenBank database under Accession Nos AJ632279-AJ632293.

Base Sequence↗

PubMed1999

Essential role of STAT3 for embryonic stem cell pluripotency.

Propagation of mouse embryonic stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated Stat3 gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the Stat3 mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the Stat3 locus, and retained normal levels of activated STAT3. Cre-regulated reduction of Stat3 gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype.

Animals↗

PubMed1999

An international collaborative study on the detection of an HIV-1 genotype B field isolate by nucleic acid amplification techniques.

An international collaborative study to assess inter-laboratory variation in the sensitivity of gene amplification assays for the detection of HIV-1 RNA sequences was conducted using a panel of eight duplicate dilutions of an HIV-1 genotype B clinical isolate and negative control samples. Twenty-five laboratories participated in the study and used a variety of in-house assays and commercial assay systems. With few exceptions, the assays were more sensitive than a p24 antigen assay. Overall, the PCR-based Amplicor Monitor assay was the most sensitive and gave the highest mean copy number for any one sample. Some of the in-house assays gave results comparable with the Monitor assay whilst the NASBA and bDNA assays appeared to be less sensitive. As a result of this study, an HIV-1 Working Reagent for the standardisation of nucleic acid amplification assays was developed and assessed in a subsequent study. Similar differences in sensitivity between the different assay systems was observed. The discrepancies in viral copy number obtained using the Working Reagent highlights the need for an International Standard against which all Working Reagents may be calibrated.

Adult↗

PubMed2002

Genetic diversity of the DBLalpha region in Plasmodium falciparum var genes among Asia-Pacific isolates.

In Plasmodium falciparum a highly polymorphic multi-copy gene family, var, encodes the variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1), which has an important role in cytoadherence and immune evasion. Using previously described universal PCR primers for the first Duffy binding-like domain (DBLalpha) of var we analysed the DBLalpha repertoires of Dd2 (originally from Thailand) and eight isolates from the Solomon Islands (n=4), Philippines (n=2), Papua New Guinea (n=1) and Africa (n=1). We found 15-32 unique DBLalpha sequence types among these isolates and estimated detectable DBLalpha repertoire sizes ranging from 33-38 to 52-57 copies per genome. Our data suggest that var gene repertoires generally consist of 40-50 copies per genome. Eighteen DBLalpha sequences appeared in more than one Asia-Pacific isolate with the number of sequences shared between any two isolates ranging from 0 to 6 (mean=2.0 +/-1.6). At the amino acid level DBLalpha sequence similarity within isolates ranged from 45.2 +/- 7.1 to 50.2 +/- 6.9%, and was not significantly different from the DBLalpha amino acid sequence similarity among isolates (P>0.1). Comparisons with published sequences also revealed little overlap among DBLalpha sequences from different regions. High DBLalpha sequence diversity and minimal overlap among these isolates suggest that the global var gene repertoire is immense, and may potentially be selected for by the host's protective immune response to the var gene products, PfEMP1.

Animals↗

PubMed2006

High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping.

Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from approximately 120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.

Cell Line, Tumor↗

PubMed1997

Analysis of the 5' upstream sequence of the Huntington's disease (HD) gene shows six new rare alleles which are unrelated to the age at onset of HD.

The CAG repeat number in the Huntington's disease (HD) gene accounts for about 50% of the variation seen in age at onset of HD. In order to determine whether promoter sequence variation can contribute to the residual variation in age at onset, we studied the conserved 303 bp region upstream of the +1 translation start site in the HD gene in a population of 56 control East Anglians, 30 Africans, 34 Japanese, and 208 English Huntington's disease patients. A surprisingly high degree of variation was found. Seven alleles were identified, comprising four polymorphisms: two single base pair substitutions, a 6 bp VNTR present as one or two copies, and a 20 bp VNTR with one to three copies of the tandem repeat. No correlation between polymorphisms and age at onset of symptoms was found in HD patients. The 6 bp and 20 bp stretches are present only in single copies in the chimpanzees and gorilla, suggesting that these VNTRs have evolved by duplication of the core sequences in the human lineage.

Age of Onset↗

PubMed2005

Evolutionary turnover of two pBuM satellite DNA subfamilies in the Drosophila buzzatii species cluster (repleta group): from alpha to alpha/beta arrays.

The pBuM satellite DNA family was studied in seven Drosophila species from the buzzatii cluster (within the large Drosophila repleta group). The pBuM repeats are slightly AT-rich and show high levels of intraspecific sequence homogeneity. The pBuM family can be divided into two subfamilies. The pBuM-1 subfamily consists of tandemly arranged repetition units of approximately 190 bp, termed alpha. Alpha repeats were found in a high copy number in the genome of D. buzzatii, D. serido and D. antonietae. The pBuM-2 subfamily consists of tandemly arranged repetition units of 370 bp. Its origin is explained by an insertion of an approximately 180 bp foreign sequence (termed beta) in an alpha basic repeat unit, with subsequent homogenization/amplification events increasing its frequency. Alpha/beta repeats were found in a high copy number in the genome of D. serido, D. antonietae, D. seriema and D. gouveai. pBuM sequences were not detected in D. koepferae and D. borborema by hybridization experiments. The nucleotide analysis of 74 pBuM repeats revealed that apart from the beta insertion event, the evolution of the pBuM family has proceeded in a gradual fashion, mainly through accumulation and horizontal spread of nucleotide substitutions. Moreover, the data also indicate a faster evolutionary rate for the pBuM-2 subfamily than the pBuM-1 subfamily. Members of both subfamilies display a greater intraspecific than interspecific homogeneity, indicating a concerted mode of pBuM evolution. A scenario to explain the evolution of both satDNA subfamilies in the seven Drosophila species from the buzzatii cluster is proposed.

Animals↗