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PubMedDate not supplied

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants.

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.

Alkylation↗

PubMed2026

An alignment-free strategy for circulating tumor DNA detection and tumor fraction estimation from whole-genome sequencing data.

Circulating tumor DNA (ctDNA) is emerging as a promising biomarker for postoperative monitoring of cancer patients. Precise estimation of circulating tumor fraction is crucial for evaluating treatment effects and timely detection of disease recurrence. All current ctDNA detection methods that utilize whole-genome sequencing (WGS) data rely on the reference genome alignment of sequencing reads and often apply separate tools for detecting different variant types. However, various bioinformatic analysis confounders and the application of external variant calling tools could be avoided by analyzing k-mers from unaligned sequencing reads. While k-mer-based methods have successfully been applied for somatic variant validation and detection, the potential of k-mer-based ctDNA detection is unexplored. We have developed a tumor-informed alignment-free ctDNA detection tool called ctDNAmer that detects tumor-specific somatic variation directly from unaligned sequencing data by identifying k-mers unique to the tumor DNA. ctDNAmer detects variant information across the genome by comparing the primary tumor and germline WGS data and accounts for sample-specific germline variability and technical noise in the same framework. We tested the utility of ctDNAmer for tumor fraction estimation on postoperative plasma cfDNA WGS data (mean sequencing depth ~ 28x) from 90 stage III colorectal cancer patients with three years of follow-up. The tumor fraction (TF) estimates agreed with the available clinical information and ctDNA was detected in 77% (17/22) of recurring patients with a median lead time of 8 months compared to radiological imaging. We further validated ctDNAmer's tumor fraction estimates based on a comparison with the mean cfDNA allele frequencies of somatic clonal SNVs identified from aligned primary tumor sequencing data. The TF estimates showed a strong Pearson correlation of 0.897 with the mean allele frequencies and improved ctDNA detection results across samples with an AUC of 0.79 compared to 0.75 if the mean allele frequency of clonal mutations is used.

Circulating Tumor DNA↗

PubMed1997

Somatic linker histones cause loss of mesodermal competence in Xenopus.

In Xenopus, cells from the animal hemisphere are competent to form mesodermal tissues from the morula through to the blastula stage. Loss of mesodermal competence at early gastrula is programmed cell-autonomously, and occurs even in single cells at the appropriate stage. To determine the mechanism by which this occurs, we have been investigating a concomitant, global change in expression of H1 linker histone subtypes. H1 histones are usually considered to be general repressors of transcription, but in Xenopus they are increasingly thought to have selective functions in transcriptional regulation. Xenopus eggs and embryos at stages before the midblastula transition are deficient in histone H1 protein, but contain an oocyte-specific variant called histone B4 or H1M. After the midblastula transition, histone B4 is progressively substituted by three somatic histone H1 variants, and replacement is complete by early neurula. Here we report that accumulation of somatic H1 protein is rate limiting for the loss of mesodermal competence. This involves selective transcriptional silencing of regulatory genes required for mesodermal differentiation pathways, like muscle, by somatic, but not maternal, H1 protein.

Activins↗

PubMed1984

Characterization of in vitro immunoselected variants from a highly metastatic murine tumor for alterations in malignant behavior in vivo.

A new Ly-6.2- antigen-loss variant (called L61 -M1) of the highly metastatic DBA/2 mouse (Ly-6.2+) MDAY-D2 tumor has been obtained by means of a monoclonal anti-Ly-6.2 antibody in an in vitro immunoselection technique. Whereas L61 -M1 grew poorly when inoculated subcutaneously into the syngeneic host, it grew and metastasized in a similar way to the parental MDAY-D2 tumor when inoculated into immunosuppressed, athymic nude mice. L61 -M1 as well as another Ly-6.2- variant of the same MDAY-D2 tumor (called L61 ) which is poorly metastatic in the syngeneic host salvaged exogenous fucose into glycoproteins and glycolipids at rates 5.5 and 7.8 times that of the parental MDAY-D2 line. In contrast, the Ly-6.2- variants exhibited a 50-70% decrease in the incorporation of exogenous mannose into glycoproteins and glycolipids. L61 -M1 and L61 also exhibited alterations in the structures of the oligosaccharide moieties linked to the cell surface glycoproteins and/or glycolipids. Thus, the in vitro immunoselection technique can be used to obtain a panel of variants with stable phenotypic alterations in their growth and metastatic capacities. Such mutants may, like previously described lectin-resistant mutants, be useful in studying the contribution of cell surface glycoproteins and glycolipids to tumorigenicity and metastasis.

Animals↗

PubMed2005

Bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease: how safe is eating beef?

Cases of bovine spongiform encephalopathy (BSE, mad cow disease) have been found in North American cattle. Its human counterpart, called variant Creutzfeldt-Jakob disease (variant CJD), is rare but seems to be linked to eating diseased beef. Many questions remain about these diseases, such as why young people seem at greater risk of variant CJD. Also, are some people more genetically at risk for acquiring variant CJD than others?

Age Factors↗

PubMed2000

Structure and expression of the variant melanin-concentrating hormone genes: only PMCHL1 is transcribed in the developing human brain and encodes a putative protein.

PMCHL1 and PMCHL2 are two copies of the so-called variant melanin-concentrating hormone (MCH) gene that are located, respectively, on human chromosome 5p14 and 5q13 and that emerged recently during primate evolution. They correspond to a 5'-end truncated version of the MCH gene mapped on chromosome 12q23 and encoding a neuropeptide precursor. The gene organization and regulation of the expression of the variant MCH genes in the human brain are the central issues we investigated. First, the structure and fine chromosomal mapping of the 5p and 5q variant MCH genes were established. These revealed several point mutations and length variations of one CA/TA repeat which allow discrimination between each copy. Using a combination of RACE-PCR, RT-PCR, and sequencing analysis, we provided strong evidence for the expression of the PMCHL1 gene but not the PMCHL2 gene in the human fetal, newborn, and adult brains. Sense, potentially coding, RNAs, as well as noncoding antisense RNAs, were identified and displayed a region-specific expression in the human brain. Strikingly, sense unspliced RNAs of the PMCHL1 gene carried a novel open reading frame and may produce an NLS-containing protein of 8 kDa named VMCH-p8. These transcripts were translated in vitro and in transfected COS cells. Therefore, the PMCHL1 gene provides a unique example of the generation of a gene in the Hominoidae lineage which is specifically transcribed in the developing human brain and has the capacity to be translated into a putative novel protein.

Amino Acid Sequence↗

PubMed1992

Promoter-selective activation domains in Oct-1 and Oct-2 direct differential activation of an snRNA and mRNA promoter.

The promoter specificity of transcriptional activators is generally thought to be conferred by the specificity of the DNA-binding domain, which brings the activation domain to the appropriate promoter sequence. We show here, however, that Oct-1 and Oct-2 can differentially activate transcription not through DNA binding specificity but instead through the use of promoter-selective activation domains. These distinct activation domains lead to stimulation of the U2 small nuclear RNA promoter by Oct-1 and an mRNA promoter by Oct-2. An Oct-2 variant, called Oct-2B, differs from Oct-2 by an Oct-1-related C-terminal extension that results from alternative splicing. This variant gains the ability to activate the U2 small nuclear RNA promoter. Thus, the promoter selectivity of a transcriptional activator can be changed, in this case by alternative splicing, without affecting its DNA binding specificity.

DNA-Binding Proteins↗

PubMed1997

Abnormal microheterogeneity detected in one commercial alpha 1-acid glycoprotein preparation using chromatography on immobilized metal affinity adsorbent and on hydroxyapatite.

The study of one commercial preparation of human alpha 1-acid glycoprotein (AAG) by isoelectric focusing and by different chromatographic methods, previously developed to purify and fractionate the genetic variants of AAG, revealed an abnormal heterogeneity for this preparation. In addition to the three main variants (F1, S and A) of AAG normally present, this preparation contained five other AAG variants (called here sigma, alpha, beta, delta and gamma), accounting for ca. 40% of the total. As it is very unlikely that the latter variants are rare AAG variants, the abnormal heterogeneity of this AAG preparation is most probably due to structural alterations occurring during the large scale isolation. The alpha and the sigma, beta, delta and gamma variants could correspond to altered forms of the A and the F1 and S variants, respectively, because of their similar retention behaviour on immobilized copper(II) ions and their similar drug binding properties. However, the elution of the variants from the immobilized metal affinity column suggested that sigma, alpha, beta, delta and gamma were desialylated. Chromatography on hydroxyapatite enabled the separation of the F1, S and A variants from the sigma, alpha, beta, delta and gamma variants. The inability of the latter variants to bind to hydroxyapatite suggests that the structural alterations might involve acidic amino acid residues. This proposal agreed with the isoelectric focusing study of variants sigma, alpha, beta, delta and gamma. Since the different separation methods used were able to resolve the variants of this AAG, this protocol could be used for characterization of commercial AAG proteins.

Animals↗

PubMed2006

Midline septa in the lumbo-sacral thecal sac: acquired abnormality or developmental anomaly? The equivalent of diastematomyelia occurring below the spinal cord?

Careful analysis of over 400 patients radiologically diagnosed with arachnoiditis showed a subgroup of eight patients with a specific set of imaging signs that suggest a pre-existing developmental abnormality mimicking the appearance of arachnoiditis. These cases have been collected over a 15-year period. No autopsy or surgical proof is available. All patients have (i) the presence of a well-defined midline septum in the sagittal plane that splits the thecal sac, usually symmetrically, in the lower lumbar or sacral region, below the level of the spinal cord; (ii) the absence of adjacent epidural fibrosis that one would expect if the septation had been caused by surgical intervention or trauma; (iii) the preservation of cerebrospinal fluid within the nerve root sheaths and around the nerve roots at the level of the septation; and in four cases (iv) the presence of either calcification or bone in this midline septum, yet nowhere else in the theca or epidural space. These cases suggest a developmental variant called, for want of a better term, 'split lumbo-sacral thecal sac' analogous to diastematomyelia but not involving the spinal cord. This variant is potentially confused with arachnoiditis or thecal scarring in the lumbo-sacral sac.

Adult↗

PubMed2004

Variant Creutzfeldt-Jakob disease and the acquired and transmissible spongiform encephalopathies.

In 1995 a fatal epidemic of spongiform encephalopathy appeared in Great Britain. The new epidemic condition was clinically and pathologically similar to Creutzfeldt-Jakob disease (CJD), a rare sporadic encephalopathy, and was called "variant CJD" (vCJD). The vCJD epidemic was detected by active epidemiologic surveillance, which had been set up in response to a British epizootic of another new disease called "bovine spongiform encephalopathy" (BSE). Widespread cattle exposure to the BSE agent (a prion) had presumably been occurring in Britain since about 1982, when changes in the rendering process allowed infected material to contaminate cattle feed. Concern that the bovine disease might transmit to humans via beef consumption led to the active surveillance program. Tragically, BSE transmission to humans appears to have led to over 100 cases of vCJD. Millions of affected cattle were eventually slaughtered throughout Europe. Cattle are now routinely tested for BSE at 24-30 months of age, and affected herds are destroyed. These measures have exponentially reduced the BSE epizootic. Britain and other European countries that received contaminated beef or cattle feed nevertheless remain concerned about the combination of nearly universal past human exposure in Britain, variable exposure elsewhere, an unknown incubation period, vCJD case totals that continue to mount, and worst-case predictions that extend to many thousands of eventual human cases.

Animals↗

PubMed1997

Temporal selectivity for complex signals by single neurons in the torus semicircularis of Pleurodema thaul (Amphibia:Leptodactylidae).

Responses of auditory neurons in the torus semicircularis (TS) of Pleurodema thaul, a leptodactylid from Chile, to synthetic stimuli having diverse temporal patterns and to digitized advertisement calls of P. thaul and three sympatric species, were recorded to investigate their temporal response selectivities. The advertisement call of this species consists of a long sequence of sound pulses (a pulse-amplitude-modulated, or PAM, signal) having a dominant frequency of about 2000 Hz. Each of the sound pulses contains intra-pulse sinusoidal-amplitude-modulations (SAMs). Synthetic stimuli consisted of six series in which the following acoustic parameters were systematically modified, one at a time: PAM rate, pulse duration, number of pulses, and intra-pulse SAM rate. The carrier frequency of these stimuli was set at the characteristic frequency (CF) of the isolated units (n = 47). Response patterns of TS units to synthetic call variants reveal different degrees of selectivities for each of the temporal variables, with populations of neurons responding maximally to specific values found in the advertisement call of this species. These selectivities are mainly shaped by neuronal responsiveness to the overall sound energy of the stimulus and by the inability of neurons to discharge to short inter-pulse gaps.

Acoustic Stimulation↗

PubMed2005

Novel insights regarding the operational characteristics and teleological purpose of the renal Na+-K+-Cl2 cotransporter (NKCC2s) splice variants.

The absorptive Na(+)-K(+)-Cl(-) cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H(2)O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H(2)O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2-NKCC2 interactions.

Alternative Splicing↗

PubMed1984

3T3 variants unable to bind epidermal growth factor cannot complement in co-culture.

A Swiss-Webster 3T3 variant, called 3T3-ENR7, unable to divide in response to epidermal growth factor was isolated by the mitogen-colchicine selection technique. Like the other EGF non-proliferative variants (3T3-NR6 and 3T3-TNR2), 3T3-ENR7 was unable to bind 125I-EGF. Pairwise co-culture of the three independently isolated EGF non-responsive variants did not restore mitogenic responsiveness to EGF.

Animals↗

PubMed1981

An examination of tumor antigen loss in spontaneous metastases.

Metastases arising from a subcutaneous injection of the DBA/2 tumor, MDAY-D2, as well as four drug-resistant variants (either wheat germ agglutinin-resistant, ouabain-resistant, or both, i.e., WGAR/OuaR) of MDAY-D2, were examined for the presence of a tumor-associated antigen (TAA). Of 15 mice examined, tumor antigen-loss variants were detected in only 1 animal. These antigen-loss metastases arose in a mouse injected with the WGAR variant called MDW4. The tumor at the site of inoculation retained the TAA, whereas all four of the metastases removed from liver, spleen and other tissues were antigen-loss variants. The antigen-loss variants were not killed by cytotoxic T cells (CTL) directed against the TAA of the parental tumor, did not competitively inhibit CTL lysis of MDW4 targets in a 'cold target' inhibition test, and were not able to elicit a CTL response. In vivo immunization-protection (challenge) experiments also showed that the metastases did not express the TAA of MDAY-D2. Unlike the WGAR phenotypes, which were lost in all spontaneous metastases recovered from MDW4-injected mice, loss of the TAA appeared to be an uncommon event. Antigen-loss tumor cell variants are discussed in terms of their relevance to metastasis, and in regard to their use in the study of T cell-mediated cytotoxicity of tumor cell populations.

Animals↗

PubMed1995

Molecular genetics of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene.

In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain, we have analysed the G6PD gene in 11 unrelated Spanish G6PD-deficient males and their relatives by using the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with a direct PCR-sequencing procedure and PCR-restriction enzyme (RE) analysis. We have identified eight different missense mutations, six of which have been reported in previously described G6PD variants. In nine patients who had presented with acute favism we found the following mutations: G6PD A-376G-202A (four cases), G6PD Union1360T (two cases), G6PD Mediterranean563T (one case) and G6PD Aures143C (one case). In the remaining patient a novel A to G transition was found at nucleotide position 209 which has not been reported in any other ethnic group. This mutation results in a (70) Tyr to Cys substitution and the resulting G6PD variant was biochemically characterized and designated as G6PD Murcia. This new mutation creates a Bsp 1286I recognition site which enabled us to rapidly detect it by PCR-RE analysis. In two patients with chronic non-spherocytic haemolytic anaemia (CNSHA) we found the underlying genetic defects, as had been noted previously, to be located within a cluster of mutations in exon 10. One of them had the T to C transition at nucleotide 1153, causing a (385) Cys to Arg substitution, previously described in G6PD Tomah. The other, previously reported as having a variant called G6PD Clinic, has a G to A transition at nucleotide 1215 that produces a (405) Met to Ile substitution, thus confirming that G6PD Clinic is a new class I variant.

Acute Disease↗

PubMed1983

Molecular and cell isoforms during development.

Development proceeds by way of a discrete yet overlapping series of biosynthetic and restructuring events that result in the continued molding of tissues and organs into highly restricted and specialized states required for adult function. Individual molecules and cells are replaced by molecular and cellular variants, called isoforms; these arise and function during embryonic development or later life. Isoforms, whether molecular or cellular, have been identified by their structural differences, which allow separation and characterization of each variant. These isoforms play a central and controlling role in the continued and dynamic remodeling that takes place during development. Descriptions of the individual phases of the orderly replacement of one isoform for another provides an experimental context in which the process of development can be better understood.

Actins↗

PubMed2006

Strategies for delaying or treating in vivo acquired resistance to trastuzumab in human breast cancer xenografts.

PURPOSE: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivo tumorigenic models. EXPERIMENTAL DESIGN: We derived an erbB-2-positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2-negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response. RESULTS: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti-epidermal growth factor receptor antibody (cetuximab) was effective as was the anti-vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors. CONCLUSIONS: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab.

Angiogenesis Inhibitors↗

PubMed1989

Molecular characterization of variant translocations in chronic myelogenous leukemia.

Five to ten percent of the Ph-positive cases of chronic myelogenous leukemia (CML), termed variant translocations, involve at least one chromosome in addition to 9 and 22 in the abnormality. The involvement of chromosome 9 band q34, where the c-abl oncogene has been localized, is not always cytogenetically detectable in so called variant translocations due to complex rearrangements. We present two cases having the most frequently involved chromosomes (#3 and #17) in such translocations. In one case, both chromosome 9's were cytogenetically normal while in the other, band 9q34 was so called 'masked' or 'hidden'. After molecular evaluation using in situ hybridization and Southern blotting techniques, the involvement of the altered bcr/abl gene was demonstrated and the cytogenetic analysis was revised. Utilization of molecular probes in the evaluation of such cases should become a routine diagnostic procedure in detecting the exchange of bcr and c-abl sequences.

Adult↗