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PubMed2002

PheGe, the platform for exploring genotype-phenotype relations on cellular and organism level.

One major challenge of bioinformatics is to extract biological information into a form that gives access to analyses and predictive models and that sheds new light on cellular and organism function. In order to approach automated network analysis on organism level the relational platform PheGe was generated. PheGe enables a) presentation of cell-specific regulatory and metabolic pathways, b) sorting and coordination of the various molecules, genes and reactions to their particular signaling systems, c) visualization of signaling par distance, d) organization of downstream events on a multi-cellular level, e) recording and evaluation of pathological relevant data, f) coordination of the aberrant genes and gene products into the various regulatory pathways balancing phenotypic patterns g) modeling of cellular differentiation and finally h) tracing of network components that balance differentiation programs.

Algorithms↗
PubMed2003

Cell phenotype analysis using a cell fluid-based microchip with high sensitivity and accurate quantitation.

We have assessed a cell fluid chip-based fluorescent cytometric assay that runs on bioanalyzer for fast characterization of small population cell phenotypes characterization. The assay determines the expression of specific cell surface markers on various cell samples. Six samples can be analyzed on each chip in one automated process. Results were in good agreement with conventional flow cytometry in quantitation. Importantly, this procedure used less than 200 cells per sample and produced results consistent with that using 10(5) cells by the conventional staining procedure. The method was also used for screening potential ingredients in herbs. Purpose of this study was to analyze the change of cell subtypes of UCB mononuclear cells in vitro reactivity in herbs. We found that by treatment of the water-soluble extract (F3) of Ganoderma lucidum, the presence of CD56(+) marker (natural killer cells) significantly increased from 1.1 to 3.2% (P<0.05 and P) in UCB mononuclear cells. The results indicated that F3 quantitatively influenced NK cells activities. We suggest this screening method may be useful for a fast phenotypes characterization after extract stimulation utilizing only a small population of cells.

Cells, Cultured↗
PubMed2006

Sequence-based typing of the complete coding sequence of DQA1 and phenotype frequencies in the Dutch Caucasian population.

Typing of DQA1 by sequencing has been a challenge because of a 3-nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but need additional exon information. With the sequencing strategy presented here the complete exons 1-4 are sequenced heterozygously, enabling identification of all DQA1 alleles by sequence-based typing (SBT). Exons 1-4 were amplified and sequenced separately, the combined sequences were used for automated allele assignment. The method was validated by typing 21 individuals with all possible different allele group combinations. In addition 26 quality control samples were correctly typed by this method. To determine the phenotype frequencies 155 unrelated Dutch Caucasian individuals were DQA1 typed. In total 15 known and two new DQA1 alleles were identified. DQA1*0103 and *0505 were the most frequent alleles with phenotype frequencies of 30% and 29%, respectively. The SBT method presented here is an improvement compared to already existing protocols in that the complete exon sequence is obtained for all coding exons, using identical polymerase chain reaction conditions. Furthermore, all exons are sequenced heterozygously, facilitating allele assignment and reducing the number of amplification reactions.

Alleles↗
PubMed2001

Facilitating haplotype analysis by fully automated analysis of all chromosomes in human-mouse hybrid cell lines.

Recent evidence suggests that haplotype analysis is essential in recognizing genetic factors involved in the tendency toward a particular disease or pharmacogenetic phenotype, as well as to identify genes involved in multigenic disorders. Because of the increasing need for efficient haplotype tests, a new hybrid system, called conversion technology, was developed. Conversion technology aims at converting the diploid chromosome content into a haploid state so that hybrids contain a single copy of any desired chromosome. A number of mutations can now be identified easily, as they are no longer obscured by the normal sequence present on the other copy of the chromosome. However, the efficient use of this hybrid system depends on a complete analysis of both human and mouse chromosome complements in order to assess the stability of the hybrid cells and to accurately determine their human chromosome content. We describe a new multicolor FISH-based method capable of analyzing both genomes simultaneously in a single hybridization. This new technique should become an instrumental part of inexpensive, reliable haplotype tests.

Animals↗
PubMed1999

Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.

BACKGROUND: The search for the eight most frequent mutations (i.e., DeltaF508, G542X, W1282X, N1303K, 1717-1G-->A, R553X, 2183AA-->G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. METHODS: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. RESULTS: The screening revealed five mutations, R1158X, 711+1G-->T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3-1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. CONCLUSIONS: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

Adult↗
PubMed2006

Short wavelength-automated perimetry compared with standard achromatic perimetry in autosomal dominant optic atrophy.

BACKGROUND: Autosomal dominant optic atrophy (ADOA, Kjer-type) is a heterogeneous, non-inflammatory degeneration of retinal ganglion cells. The diagnosis of ADOA can be challenging owing to its insidious onset and large variability in phenotypic expression, both within and between individual pedigrees. The earliest literature reports relatively mild centrocaecal scatomas to white targets in ADOA, but extensive and dense peripheral field loss to coloured targets, especially blue, with Bjerrum perimetry. The phrase "inverted peripheral visual fields to coloured targets" has been used to describe this phenomenon. METHODS: Humphrey standard achromatic perimetry (SAP) and short wavelength-automated perimetry (SWAP) were carried out on five patients with ADOA. RESULTS: Regardless of wide variations in patient age, visual acuity, disc appearance and colour vision, the SWAP mean deviation (MD) was between 10 and 20 times more depressed than the SAP MD. The actual differences ranged from 9.38 to 13.78 dB. CONCLUSIONS: These data are consistent with the original reports suggesting that, early in this disease process, the blue-target deficits are typically peripheral and that this difference between SAP and SWAP perimetry may be a robust indicator of ADOA in both early and late stages of this disease.

Adolescent↗
PubMed2006

On the difference of micronucleus frequencies in peripheral blood lymphocytes between breast cancer patients and controls.

Sporadic breast cancer patients as well as mutation carriers of the BRCA genes have a reduced DNA repair capacity compared to controls when assessed by the G0 micronucleus test (G(0)MNT) or by induced chromosomal aberrations. Since the individual MN frequencies vary widely and overlap between cases and controls it remained unclear which percentage of the cases should be considered to exhibit an increased radiosensitivity. We performed a similar case-control study and found a highly significant difference (P < 0.0001) between all breast cancer cases (N = 91) and female controls (N = 96) using descriptive statistics and ANOVA with adjustment for age. This difference also holds for baseline MN frequencies (P = 0.0006) and for subgroups of the patients similar to those without treatment (P < 0.0001). These results were confirmed in a second sample acquired at a different hospital. Since we are dealing in this analysis with two predefined groups (patients and controls), we calculated odds ratios (ORs) in order to assess the discriminative power of the G0 MNT. These amounted to OR = 4.9 (P < 0.0001) for MN frequencies obtained by visual counting and ranged from OR = 11 (P < 0.0011) to OR = 22 (P < 0.0001) using automated counting. In order to overcome the problem of choosing a cut-off point inherent in ORs, receiver operating characteristic curves were calculated, which visualize specificity and sensitivity over the entire range of values and which characterize the discriminative power of a test by the area under the curve (AUC) (visual counting, baseline: AUC = 0.67; induced AUC = 0.75; automated counting: AUC > 0.88; evaluation sample: AUC > 0.73). We conclude that the G0 MNT may be a useful tool to substitute the phenotype breast cancer in association and linkage studies and that it may be possible to develop a test useful in the diagnosis or risk assessment for breast cancer.

Adult↗
PubMed1991

[Characterization and validation of a human anti-C monoclonal antibody].

Peripheral lymphocytes were obtained from an immunized woman against C and Ce antigens. Punction was realized 14 days after childbirth. After hetero-hybridization, a unique cell line continued secreting a monoclonal IgM antibody. Serological characterization of this antibody was determined by direct agglutination tests against 150 native and enzyme treated red blood cells including some rare phenotypes. This antibody was specific of C determinant of the Rh system. It showed strong reactions by saline and enzymatic technics, against C positive cells from Ce positive and Ce negative patients. The validation was performed by a manual direct agglutination test in saline (tube) and by an automated-hemagglutination test (in microplate) against 2,500 patients samples. No discrepancy was observed. This monoclonal IgM anti-C could be used as a potent reagent and since one year, about 30,000 patients and pregnant women have been phenotyped in our laboratory successfully.

Adult↗
PubMed2000

Identification of immunological relevant phenotypes in ENU mutagenized mice.

The immunology screen focuses on the identification of novel gene products involved in the mammalian immune response and on the establishment of mouse models for immunological disorders. For this purpose, high throughput and semi-automated techniques were developed and optimized for low cost per sample and reproducibility. All assays are designed to be nonconsumptive and are based on peripheral blood or direct PCR amplification.

Animals↗
PubMed2005

Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry.

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false-positive and false-negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild-type protein expressed in normal cells, such that homozygous gene deletion or non-sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post-translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21(WAF1) and MDM2, and statistical correlations between MDM2/p21(WAF1) and p53 were found in tumours with wild-type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

Adenocarcinoma↗
PubMed2004

Determination of epidemic clonality among multidrug-resistant strains of Acinetobacter spp. and Pseudomonas aeruginosa in the MYSTIC Programme (USA, 1999-2003).

The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Programme was initiated in 1997 (1999 for the United States). This program monitors resistance in participant medical centers where carbapenems are prescribed and drug use data can be obtained. An earlier report found antimicrobial use was not a clear cause of local or aggregate changes in resistance rates. This study addresses the role of dissemination of resistant clones on susceptibility rates for nonfermentors, Acinetobacter spp. (ACB) and Pseudomonas aeruginosa (PSA). Carbapenem (CARB)-multidrug-resistant strains (MDR) from among 236 ACB and 1,111 PSA were tested by reference broth microdilution methods, automated ribotyping, and pulsed field gel electrophoresis to determine possible clonal dissemination. Each strain was also tested for metallo-beta-lactamases (MbetaL) (phenotypic and polymerase chain reaction); and then analyzed by CARB-R rate and defined daily dose (DDD)/100 days use groupings (high, moderate, and low). For the aggregate 15 sites in the MYSTIC Programme each year, the CARB-resistant rate decreased over 5 years; but other drug-resistance rates generally escalated. Changes were not related to antimicrobial use calculations. The discovered clonally spread MDR-PSA strains were more frequent in high- (1.8 clones/site) and moderate-resistance (0.6 clones/site) rate centers (21.7% to 29.5% were clonal), compared with unique strains in low-resistance hospitals. ACB clonality was extreme in one geographic area, with dissemination of 5 different clones (931.7/B, C, or D; 1090.2/A; 167.5/A) in 4 centers (02, 04, 06, and 18). Resistance rates in ACB and PSA were clearly related to clonal occurrence and spread, and one MbetaL (VIM-7) was detected. Decreased CARB resistance rates from 1999 through 2002 were directly attributed to the disappearance of resistance clones in some locations. In conclusion, ACB and PSA CARB and MDR resistance rates in MYSTIC Programme institutions have been greatly influenced by clonal dissemination and less by antimicrobial use patterns. The most serious examples of resistance were the clonality observed among ACB in New York City and the documented endemic nature of VIM-7-producing PSA (0.09% of all PSA isolates). Meropenem remained the most active antimicrobial agent tested in the program, and surveillance networks must implement epidemiologic typing to accurately assess the role of clonal spread on the study results.

Acinetobacter↗
PubMed1996

Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis.

Flow cytometry has become the preferred method for the lineage assignment and maturational analysis of malignant cells in acute leukemias and lymphomas. Multiparametric immunophenotyping allows the detection of aberrant antigen coexpression and the analysis of heterogeneity and clonality of malignant cells in leukemias and lymphomas. The complexity of multiparameter analysis techniques and the multitude of available monoclonal antibodies demand a standardization of protocols for the use of flow cytometry in clinical laboratories in order to achieve interlaboratory reproducibility. Therefore, the Working Group on Flow Cytometry and Image Analysis has started an initiative in order to establish a consensus protocol on the current methods of the phenotyping of hematological neoplasias as a basis for quality assurance and support for upcoming technologies such as quantitative analysis of antigen densities and automated knowledge-based analysis software. In addition to general recommendations on assay procedures and quality control specific recommendations are given for the selection of two-color reagent panels and data interpretation in an attempt to define a basis for cross-evaluation against the different currently established laboratory protocols.

Acute Disease↗
PubMed1994

A method for detecting DNA polymorphism in large populations.

Molecular markers linked to loci of interest can be used for fine mapping a particular area of a genome or for marker-assisted selection. We present an approach for screening individual plants with polymorphic markers that facilitates phenotyping in large populations. Polymorphic DNA fragments, amplified by PCR, are labelled with digoxigenin and used as probes on slot blots of amplified DNA from the individual plants to be tested. DNA is obtained by a simple two-tube purification method. The colorimetric detection of alleles on the blots is more reliable, and more amenable to automation, than conventional staining of electrophoresis gels.

DNA↗
PubMed2005

Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets.

Forward Pharmacology seeks to identify small or large molecules that modulate a normal or abnormal biological process in living cells or whole organisms and historically has been responsible for the discovery of many clinically used drugs. Forward Pharmacology approaches have become particularly attractive because advances in combinatorial chemistry and laboratory automation have made it possible to generate and interrogate large compound collections in a short period of time. Because many drug discovery efforts are now directed against specific biochemical targets, however, the utility of Forward Pharmacology is limited by the fact that assays to investigate compounds in biological systems are often phenotypic rather than target specific. We discuss here a novel strategy to discover target-based small molecules in intact cells using contemporary Forward Pharmacology in cells with specific genetic manipulations. The method, which we have termed "chemical complementation", is defined as the ability of small molecules to reverse a genetically induced phenotypic change in intact cells. Chemical complementation represents an extension of the commonly used genetic complementation approach, where cDNA libraries are used to investigate the function of genes based on their ability to rescue a specific genetic defect. We present examples of how chemical complementation has been used to identify and credential cell-active, small molecule inhibitors of 2 dual-specificity phosphatases, Cdc25A and MKP-3, which heretofore have eluded small molecule drug discovery efforts.

Animals↗
PubMed2006

Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.

Benzimidazoles↗
PubMed1998

Genotype-phenotype correspondence in Sanfilippo syndrome type B.

Sanfilippo syndrome type B, or mucopolysaccharidosis type IIIB, results from defects in the gene for alpha-N-acetylglucosaminidase (NAGLU); only a few mutations have been described. To rapidly identify most NAGLU mutations, an automated sequencing procedure was developed for analysis of the entire coding region, including exon-intron borders. By this method, eight affected families were studied, and the mutations in all 16 alleles were identified, more than doubling the number of published mutations for this gene. Eight mutations were described for the first time: five missense mutations (Y140C, Y455C, P521L, S612G, and R674C), two nonsense mutations (W675X and Q706X), and one 24-nucleotide insertion. Currently, 36% of all point mutations (8 of 22 alleles) involve R674, a codon having a CpG dinucleotide in the critical initial position. Other mutations were found in more than one family, raising the possibility that some may be relatively common and, possibly, ancient mutations. Six new nonpathological mutations were also identified and likely represent polymorphic variants of the NAGLU gene, two of which might alter enzyme level. Establishing genotype-phenotype relationships will be vital in the evaluation of experimental treatments such as gene therapy.

Acetylglucosaminidase↗
PubMed2003

Extended blood grouping of blood donors with automatable PCR-ELISA genotyping.

BACKGROUND: In the past 10 years, PCR-based methods have been described to allow the detection of gene polymorphisms responsible for many blood group antigens. These methods are routinely used to test samples of fetal origin and to resolve serologic discrepancies. Another interesting application of blood group genotyping could be the extended typing of blood donors for minor antigens to facilitate the procurement of compatible blood for alloimmunized patients. STUDY DESIGN AND METHODS: PCR-based tests have been modified to allow multiplex amplification of specific fragments of blood group genes and the convenient detection of hybridized amplicons by ELISA in a microplate format. RESULTS: The results obtained show that fragments of the Rh (D, c, C, e, E), Kell (K, k), Duffy (Fya, Fyb), and Kidd (Jka, Jkb) genes could be amplified along with controls in multiplex PCR reactions. Labeling of amplicons with digoxigenin allowed their solid-phase detection in microplate wells previously coated with individual blood group-specific oligonucleotides. A comparative study performed with 100 individuals showed a 99.7 percent concordance between genotypes and phenotypes for the 11 antigens assayed, with only three discrepant Fyb genotypes. CONCLUSION: Extended genotyping could be performed once on regular donors and confirmed when needed by standard serologic RBC assays. The format of these tests will allow easy automation of the procedure including the interpretation and downloading of the results with existing ELISA software.

Automation↗