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PubMed2006

Transcriptome profiling in clinical breast cancer: from 3D culture models to prognostic signatures.

Early detection has been one of the most effective strategies to control the growing cancer burden. The power of earlier detection has been demonstrated by the impact of pap-smear, mammography, and PSA tests on cancer patient treatment and survival. These tests benefit patients independent of their genetic background or race. However, in many cases, we are still losing the battle against cancer because patients that initially presented with low-grade disease progress rapidly to aggressive forms of the disease. As of yet, we have limited means to predict a particular patient's fate or to specifically treat subtypes of cancer. A combination of earlier detection and targeted therapy, based on information from transcriptome analysis, could be a powerful ally in this battle. The theme of this review article is to briefly summarize innovative strategies using three-dimensional (3D) cell cultures of human mammary epithelial cells to predict clinical outcome in breast cancer. This strategy has the potential to further enhance our understanding of breast cancer biology and to contribute to the identification of biologically significant bio-markers that are also useful drug targets.

Biomarkers, Tumor↗

PubMed1989

Genetic influences on behavior of infant Mus domesticus: a comparison of results from diallels derived from single and multiple populations.

A complete diallel cross was generated from six Jax inbred strains of Mus domesticus from diverse origins and a second 6 x 6 diallel generated from strains derived from a single wild population. During their second day of life, infants from both diallels were tested for latency to orient toward and root beneath mothers and, in a separate test, for latency to attach to mother's nipple. Rooting latency showed a significant additive maternal strain effect but little systematic effect of pup genotype. Nipple attachment latencies exhibited complete genetic dominance favoring rapid attachment, with no maternal effects. Patterns of genetic and influences obtained from the two diallels were highly similar for both behaviors, suggesting that for many traits the requirement that strains be drawn from a common base population may be relaxed.

Animals↗

PubMed1990

Amylase-binding as a discriminator among oral streptococci.

The ability of 51 strains, belonging to Streptococcus sanguis, 'S. mitior', S. oralis and related groups, to bind salivary amylase was studied. Most strains were grouped according to their DNA-relatedness and then compared using 14 phenotypic tests. S. mitis, 'S. mitior' and three relatively new groups of strains ('CR', 'MGH' and 'Tufted mitior') bound salivary amylase, while strains of S. sanguis and S. oralis did not. The ability of strains to bind amylase or not was remarkably consistent within groups and the test proved to be reproducible, rapid and easy to perform. Combination of the amylase-binding test with 6 other conventional physiological tests allowed the construction of a dichotomous identification key which correctly identified 95% of strains for which genetic data was available. These findings suggest that the ability of organisms to bind salivary amylase could become a key test in identification schemes for certain oral streptococci.

DNA, Bacterial↗

PubMed2006

Identification of a canine model of pyruvate dehydrogenase phosphatase 1 deficiency.

Exercise intolerance syndromes are well known to be associated with inborn errors of metabolism affecting glycolysis (phosphorylase and phosphofructokinase deficiency) and fatty acid oxidation (palmitoyl carnitine transferase deficiency). We have identified a canine model for profound exercise intolerance caused by a deficit in PDP1 (EC 3.1.3.43), the phosphatase enzyme that activates the pyruvate dehydrogenase complex (PDHc). The Clumber spaniel breed was originated in 1760 by the Duc de Noailles, as a hunting dog with a gentle temperament suitable for the 'elderly gentleman'. Here we report that 20% of the current Clumber and Sussex spaniel population are carriers for a null mutation in PDP1, and that homozygosity produces severe exercise intolerance. Human pyruvate dehydrogenase phosphatase deficiency was recently characterized at the molecular level. However, the nature of the human mutation (loss of a single amino acid altering PDP1 activity) made it impossible to discern the role of the second phosphatase isoform, PDP2, in the deficient phenotype. Here we show that the null mutation in dogs provides a valuable animal model with which to study the effects of dysregulation of the PDHc. Knowledge of the molecular defect has allowed for the institution of a rapid restriction enzyme test for the canine mutation that will allow for selective breeding and has led to a suggested dietary therapy for affected dogs that has proven to be beneficial. Pharmacological and genetic therapies for PDP1 deficiency can now be investigated and the role of PDP2 can be fully characterized.

Animals↗

PubMed1999

Enhancement of bleomycin-induced micronucleus formation in V79 cells as a rapid and sensitive screen for non-covalent DNA-binding compounds.

Non-covalent drug/DNA interactions are difficult to study and because of this, the significance of such interactions from a safety standpoint and their contribution to positive genetic toxicology test findings is poorly understood. It is shown in the present study that such interactions may be detected and quantified in Chinese hamster V79 cells by an adaptation of the bleomycin amplification assay. This assay measures the ability of a test compound to enhance the DNA damaging activity of the antibiotic bleomycin using micronucleus formation as an endpoint. Results are presented examining the bleomycin amplification activity of known intercalating agents, groove-binding agents and other structurally diverse classes of compounds for which intercalative status has not been reported. The assay reveals a strong and predictable SAR for amplification activity based on number and orientation of aromatic rings. Moreover, excellent correlations are observed between DNA binding (viscometric analyses) and DNA amplification in V79 cells for a series of seven experimental compounds. The assay is shown to be useful in understanding the genotoxicity of marketed antihistamines and to help explain genetic toxicology findings observed in a series of novel pharmaceutical entities. It is proposed that assessment of bleomycin amplification activity of novel compounds in early genotoxicity prescreening may provide important information upon which to base synthesis of compounds with minimal or no genotoxic liability.

Animals↗

PubMed2001

Replication validity of genetic association studies.

The rapid growth of human genetics creates countless opportunities for studies of disease association. Given the number of potentially identifiable genetic markers and the multitude of clinical outcomes to which these may be linked, the testing and validation of statistical hypotheses in genetic epidemiology is a task of unprecedented scale. Meta-analysis provides a quantitative approach for combining the results of various studies on the same topic, and for estimating and explaining their diversity. Here, we have evaluated by meta-analysis 370 studies addressing 36 genetic associations for various outcomes of disease. We show that significant between-study heterogeneity (diversity) is frequent, and that the results of the first study correlate only modestly with subsequent research on the same association. The first study often suggests a stronger genetic effect than is found by subsequent studies. Both bias and genuine population diversity might explain why early association studies tend to overestimate the disease protection or predisposition conferred by a genetic polymorphism. We conclude that a systematic meta-analytic approach may assist in estimating population-wide effects of genetic risk factors in human disease.

Bias↗

PubMed1998

Gyrase and Topo IV modulate chromosome domain size in vivo.

In bacteria, DNA supercoil movement is restricted to subchromosomal regions or 'domains.' To elucidate the nature of domain boundaries, we analysed reaction kinetics for gammadelta site-specific resolution in six chromosomal intervals ranging in size from 14 to 90 kb. In stationary cultures of Salmonella typhimurium, resolution kinetics were rapid for both short and long intervals, suggesting that random stationary barriers occur with a 30% probability at approximately 80 kb intervals along DNA. To test the biochemical nature of domain barriers, a genetic screen was used to look for mutants with small domains. Rare temperature-sensitive alleles of DNA gyrase and Topo IV (the two essential type II topoisomerases) had more supercoil barriers than wild-type strains in all growth states. The most severe gyrase mutants were found to have twice as many barriers in growing cells as wild type throughout a 90 kb interval of the chromosome. We propose that knots and tangles in duplex DNA restrain supercoil diffusion in living bacteria.

Chromosomes, Bacterial↗

PubMed1996

Virulence factors of medically important fungi.

Human fungal pathogens have become an increasingly important medical problem with the explosion in the number of immunocompromised patients as a result of cancer, steroid therapy, chemotherapy, and AIDS. Additionally, the globalization of travel and expansion of humankind into previously undisturbed habitats have led to the reemergence of old fungi and new exposure to previously undescribed fungi. Until recently, relatively little was known about virulence factors for the medically important fungi. With the advent of molecular genetics, rapid progress has now been made in understanding the basis of pathogenicity for organisms such as Aspergillus species and Cryptococcus neoformans. The twin technologies of genetic transformation and "knockout" deletion construction allowed for genetic tests of virulence factors in these organisms. Such knowledge will prove invaluable for the rational design of antifungal therapies. Putative virulence factors and attributes are reviewed for Aspergillus species, C. neoformans, the dimorphic fungal pathogens, and others, with a focus upon a molecular genetic approach. Candida species are excluded from coverage, having been the subject of numerous recent reviews. This growing body of knowledge about fungal pathogens and their virulence factors will significantly aid efforts to treat the serious diseases they cause.

Antigens, Fungal↗

PubMed1998

[Histopathologic consultations for quality control. Ethical aspects].

Histopathology and cytopathology are subjective, descriptive methods and there is a need for second opinion of second pathologist. This should be solved in a systematic way from both, organisational and financial point o view. This could not rely on a friendly consultation of a colleague pathologist only. Consultations are integral part of quality assurance and quality control. Quality control in gynaecological cytopathology is associated with reexamination of 1 in every 100 negative cytological smears. High grade dysplasia of the gastric mucosa requires rapid second endoscopy and second opinion prior to gastrectomy. All this is discussed from the point of view of common sense and ethics which (thanks God) are compatible. Protection against unwanted genetic molecular tests still is not satisfactory.

Ethics, Medical↗

PubMed1979

Mapping chromosomal genes of Saccharomyces cerevisiae using an improved genetic mapping method.

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + approximately n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and alpha strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.

Aneuploidy↗

PubMed2001

Rapid molecular analysis of the haemagglutinin gene of human influenza A H3N2 viruses isolated in spain from 1996 to 2000.

A simple molecular technique was used for the rapid preliminary genetic characterization of human influenza A H3N2 viruses isolated in Spain from 1996 to 2000. Subtyping, based on RT-PCR, was followed by subtype-specific restriction enzyme fragment length polymorphism (RFLP) analyses of an amplified region of the HA1 domain of the H3 haemagglutinin (HA) gene to distinguish variants differentiated by common amino acid substitutions in HA1. The approach was tested using 135 Spanish H3N2 isolates and included nucleotide sequencing and phylogenetic analyses of a region of the HA1 domain of 41 representative isolates. The viruses were distinguished by haemagglutination inhibition (HI) assays into two antigenically discernible groups, the A/Wuhan/359/95-like and A/Sydney/5/97-like viruses. The results of PCR-RFLP analysis allowed a finer classification into five genetic variant subgroups, corresponding to those distinguished by phylogenetic analyses. This rapid, simple and variant-specific procedure could, therefore, be used to rapidly screen clinical specimens prior to more detailed antigenic and genetic analyses.

Hemagglutinin Glycoproteins, Influenza Virus↗

PubMed2002

Parental genetic contributions in the AXB and BXA recombinant inbred mouse strains.

Recombinant inbred (RI) strains are a valuable tool in mouse genetics to rapidly map the location of a new locus. Because RI strains have been typed for hundreds of genetic markers, the genotypes of individual strains within an RI set can be examined to identify specific strain(s) containing the desired region(s) of interest (e.g., one or more quantitative trait loci, QTLs) for subsequent phenotype testing. Specific RI strains might also be identified for use as progenitors in the construction of consomic (chromosome substitution strains or CSSs) or congenic lines or for use in the RI strain test (RIST). To quickly identify the genetic contributions of the parental A/J (A) and C57BL/6J (B) strains, we have generated chromosome maps for each commercially available AXB and BXA RI strain, in which the genetic loci are color-coded to signify the parent of origin. To further assist in strain selection for further breeding schemes, the percentages of A and B parental contributions were calculated, based on the total number of typed markers in the database for each strain. With these data, one can rapidly select the RI strain(s) carrying the desired donor and recipient strain region(s). Because points of recombination are known, starting with RI mice to generate CSSs or congenic lines immediately reduces genomewide screening to those donor-strain regions not already homozygous in the recipient strain. Two examples are presented to demonstrate potential uses of the generated chromosome maps: to select RI strains to construct congenic lines and to perform an RIST for Aliq1, a QTL linked to ozone-induced acute lung injury survival.

Animals↗

PubMed1997

[Rapid genetic diagnosis of females carriers related to patients with choroideremia].

PURPOSE: By using the single strand conformation analysis to search for point mutations in the choroideremia gene, we had previously identified the first truncative mutation responsible for CHM in France. The aim of the present study was to perform a simple and nonisotopic routine test to identify carriers and non carriers in the relevant family. METHODS: We used a PCR-based restriction analysis to detect the presence or absence of the mutation in the family members, as the mutation creates a restriction site in the coding sequence of the CHM gene. RESULTS: We could follow the segregation of the mutation in the pedigree, and unambiguously determine the genetic status of the females. CONCLUSION: When a mutation responsible for choroideremia modifies a restriction site, the PCR-restriction provides an efficient and unexpensive one-day test to detect heterozygosity in the family.

Carrier State↗

PubMed2000

Psychological consequences of predictive genetic testing: a systematic review.

The aim of this systematic literature review is to describe the psychological consequences of predictive genetic testing. Five databases were searched for studies using standardised outcome measures and statistical comparison of groups. Studies were selected and coded by two independent researchers. From 899 abstracts, 15 papers, describing 11 data sets, met the selection criteria for the review. The studies were of predictive genetic testing for Huntington's disease, hereditary breast and ovarian cancer, familial adenomatous polyposis and spinocerebellar ataxia. One involved children; the rest were of adults. None of the 15 papers reported increased distress (general and situational distress, anxiety and depression) in carriers or non-carriers at any point during the 12 months after testing. Both carriers and non-carriers showed decreased distress after testing; this was greater and more rapid amongst non-carriers. Test result (ie being a carrier or non-carrier) was rarely predictive of distress more than one month after testing (predictive in two of 14 analyses). Pre-test emotional state was predictive of subsequent distress in 14 of 27 analyses. There is a lack of informative studies in this field. The studies reviewed suggest that those undergoing predictive genetic testing do not experience adverse psychological consequences. However, the studies are of self-selected populations who have agreed to participate in psychological studies and have been followed up for no more than three years. Most research has been of testing for Huntington's Disease and included follow-up of no more than one year. The results suggest that testing protocols should include a pre-test assessment of emotional state so that post-test counselling can be targeted at those more distressed before testing. None of the studies experimentally manipulated the amount or type of counselling provided. The relationship between counselling and emotional outcome is therefore unclear and awaits empirical study.

Adaptation, Psychological↗

PubMed1980

Whole-mammal mutagenicity tests: evaluation of five methods.

Transmitted genetic changes in mammals can be used to study all of the main endpoints of mutagenesis: point mutations, chromosome breakage, with or without rearrangement, and chromosome nondisjunction. Four methods most commonly employed in whole-mammal germ-line mutagenicity tests as well as an in vivo somatic prescreen, are summarized. Genetic basis, historical background, description of the test, limitations, and strengths are presented for each of the five systems. The specific-locus test using visible markers is the most reliable and practical method for detecting heritable point mutations, including small deficiencies, and does not require very large numbers of animals for risk extrapolations, if a relatively high dose can be administered without killing germ cells. For the detection of chromosome-breakage events, dominant lethals are useful to determine relative sensitivities of different germ-cell stages of the male, but the heritable-translocation test is more sensitive when the exposed cells are male meiotic and postmeiotic stages. Chromosome breakage events in the female are best revealed through a sex-chromosome loss test, which utilizes genetically marked X chromosomes. The same method can also be employed to detect nondisjunction in either sex. The in vivo somatic mutation test is useful as a prescreen for both point mutations and losses of chromosomal material. The reliability and efficiency of whole-mammal mutagenicity tests must be considered in two contexts: in the assessment of mutagenicity per se (as this applies to genetic changes transmitted to future generations), and in the use of mutagenicity as a possible indicator of carcinogenicity. In the former context, the whole-mammal tests are irreplaceable because they provide the closest practicable approach to the metabolic pathways existing in man, and because there is no array of lower-system tests that can predict the complexity of the response of the different mammalian germ-cell stages (which differ greatly with respect both to their absolute and their relative sensitivities to the induction of various genetic endpoints). In the second context, i.e., the use of mutagenicity as a screen for carcinogenicity in the exposed individual, most of the whole-mammal tests are of more limited utility, because they require several weeks or months for completion. Of the methods discussed, the spot test appears most suitable, because it provides a relatively rapid in vivo system capable of detecting both gene mutations and chromosomal changes of various kinds in somatic cells, including some that have been suggested to be involved in tumor promotion.

Animals↗

PubMedDate not supplied

Cystic fibrosis carrier screening: validation of a novel method using BeadChip technology.

PURPOSE: To validate a novel BeadChip assay system for cystic fibrosis (CF) mutation testing using the panel of 25 ACMG recommended mutations and D1152H. METHODS: DNA from 519 individuals originally tested for CF mutation status by allele specific oligonucleotide hybridization (ASOH) were blindly analyzed by the BeadChip assay and the results were compared. The elongation mediated multiplexed analysis of polymorphisms (eMAP) protocol, which combines multiplex amplification of genomic DNA and multiplex detection of mutations on color-coded bead arrays, was used to analyze 26 CF mutations in two separate groups. RESULTS: The system accurately distinguished the 26 CF genotypes and had 100% concordance with the ASOH technique with an assay failure rate of 1.7%. Benign variants of exon 10 codons 506, 507, and 508 did not interfere with mutation identification and reflex testing for the 5/7/9T IVS8 polymorphism was performed on a separate array. CONCLUSIONS: The BeadChip assay system provided accurate and rapid identification of the ACMG recommended CF mutations.

Cystic Fibrosis↗

PubMed2005

Molecular genetic confirmatory testing from newborn screening samples for the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations.

beta-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with beta-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for beta-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for beta-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (-88 and -29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, -28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these beta-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with beta-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific beta-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation.

Asian People↗

PubMed2005

Simultaneous detection of isoniazid, rifampin, and ethambutol resistance of Mycobacterium tuberculosis by a single multiplex allele-specific polymerase chain reaction (PCR) assay.

Prompt detection of drug resistance of Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a multiplex allele-specific polymerase chain reaction (MAS-PCR) that detects the most commonly observed isoniazid (INH), rifampin (RIF), and ethambutol resistance-associated mutations in a single assay. The usefulness of the newly developed method was evaluated with 174 clinical isolates of M. tuberculosis obtained from Turkey. Distinct PCR banding patterns were observed for different mutation profiles and the correlation between MAS-PCR results and DNA sequencing findings was 99.4%. With culture-based phenotypic drug susceptibility testing as a reference standard, the sensitivity and specificity of the newly developed MAS-PCR assay for drug resistance-related genetic mutation detection were determined to be 81.1% and 97.5% for INH, 93.0% and 98.9 % for RIF, and 54.5% and 68.0 % for ethambutol. MAS-PCR provides a rapid, potentially more cost-effective, method of detecting multidrug-resistant TB.

Alleles↗