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Results for “Automated phenotyping”

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PubMed2005

Detecting mismatch repair defects in myeloma.

Defects in the mismatch repair system are associated with a microsatellite unstable phenotype. In this chapter, we describe the preparation of purified plasma cells using CD138 magnetic microbeads as a source of tumor DNA. We also describe a robust, sensitive method for comparing microsatellite repeat units of tumor to constitutive DNA using polymerase chain reaction and laser scanning of fluorescently labeled amplicons in an automated sequencer in order to assess microsatellite instability in myeloma.

Antigens, CD↗

PubMed2005

A systematic high-throughput screen of a yeast deletion collection for mutants defective in PHO5 regulation.

In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. One of these genes is PHO5, which encodes a secreted acid phosphatase. A phosphate-responsive signal transduction pathway (the PHO pathway) mediates this response through three central components: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. While signaling downstream of the Pho81/Pho80/Pho85 complex to PHO5 expression has been well characterized, little is known about factors acting upstream of these components. To identify missing factors involved in the PHO pathway, we carried out a high-throughput, quantitative enzymatic screen of a yeast deletion collection, searching for novel mutants defective in expression of PHO5. As a result of this study, we have identified at least nine genes that were previously not known to regulate PHO5 expression. The functional diversity of these genes suggests that the PHO pathway is networked with other important cellular signaling pathways. Among these genes, ADK1 and ADO1, encoding an adenylate kinase and an adenosine kinase, respectively, negatively regulate PHO5 expression and appear to function upstream of PHO81.

Acid Phosphatase↗

PubMed2002

Mutations in the von Hippel-Lindau (VHL) gene refine differential diagnostic criteria in renal cell carcinoma.

BACKGROUND AND OBJECTIVES: Renal cell carcinomas (RCC) with abundant granular cytoplasm include oncocytomas, eosinophilic variants of chromophobe RCC, papillary RCC, collecting duct carcinoma, and some conventional (clear cell) RCC. Tumors with predominantly clear cell cytoplasm include typical chromophobe RCC and conventional (clear cell) RCC. The objective of this study was to determine if mutations in the VHL gene can serve as auxiliary diagnostic criteria in refining histology based subtyping of renal epithelial neoplasia. METHODS: The study cohort of 67 cases included 24 conventional RCC, 14 chromophobe RCC, 14 papillary RCC, and 15 oncocytomas. Single strand conformational polymorphism (SSCP) was used as a screening procedure for mutations followed by automated sequencing to identify mutations. RESULTS: Thirteen of the 14 mutations identified were novel, seven of which were in the coding region. In chromophobe RCC, mutations clustered in the 5'UTR/promoter region and have not been previously reported. Exon 3 appeared to favor conventional (clear cell) RCC and correlated with a more aggressive phenotype. Mutations were absent in the papillary and oncocytoma RCC subtypes. CONCLUSIONS: Exon 3 mutations permitted a morphological distinction between conventional (clear cell) RCC and chromophobe RCC with clear cells. Mutations in the VHL gene refine histologic diagnostic criteria in RCC serving as adjuncts to the present morphology based diagnosis of RCC.

Adult↗

PubMed1999

Leucocyte activation markers in clinical practice.

The response against tissue injury and infection begins with the early activation of molecular and cellular elements of the inflammatory and immune response. Severe tissue injury, necrosis, and infection induce imbalanced inflammation associated with leukocyte over-stimulation and excessive or dysregulated release of cellular mediators. Clinical and experimental studies have shown that these mediators are directly related to progressive post-injury complications. Persistent increased levels of pro-inflammatory mediators produce tissue injury. Excessive production and activity of anti-inflammatory mediators cause anergy and/or immune dysfunction with increased susceptibility to infection. Leukocyte activation is assessed by cell surface phenotype expression, cellular mediators determination, or by measuring functional responses using isolated cells. Potential routine clinical uses are: evaluation of severity and prognosis in critically ill patients, immunomonitoring of sepsis, and detection of tissue injury, necrosis, and infection. In practice, the determination of cellular activation markers is restricted by a limited number of automated methods and by the cost of reagents. The availability of flow cytometry and immunoassay automated systems can contribute to a wider use in practice. Here we review the immunopathophysiology of polymorphonuclear neutrophil, monocyte, macrophage, and lymphocyte activation in response to tissue injury and infection. In addition, laboratory methods for their determination, and clinical applications in practice, are discussed.

Animals↗

PubMed1983

Obstetric care of the affected carrier of hemophilia B.

Hemophilia B (factor IX deficiency) is an X-linked recessive clotting disorder similar to hemophilia A. In both diseases, phenotypic expression of the disorder may occur in significant numbers of heterozygous carriers. This is presumably due to random inactivation of one of the two X chromosomes of the female carrier as described by the Lyon hypothesis. Described herein is the obstetric care of a severely affected heterozygous carrier of hemophilia B. Predelivery automated plasma exchange was used to raise factor IX levels successfully. Commercial factor concentrates were avoided. The Lyon hypothesis is discussed in detail, and recommendations are made for the care of the clotting factor-deficient gravid woman.

Adult↗

PubMed1998

Bacterial tracking in a dairy production system using phenotypic and ribotyping methods.

A systematic sampling plan was designed to collect raw and pasteurized milk samples throughout a single-raw milk source, dairy-processing operation experiencing reduced product shelf lives due to bacterial contamination. The objectives were to track bacterial contamination sources throughout a complete dairy production system and use this information to reduce bacterial spoilage losses in processed fluid products. Over a 5-week period, 233 bacterial isolates were collected, representative of different colony morphologies on psychrotrophic bacteria count (PBC) plates. Forty-five isolates (19%) were obtained from pasteurized milk and 188 (81%) were isolated from raw product. Thirty isolates were identified as Pseudomonas spp. by Gram stain and biochemical methods. Of these, 27 (90%) were postpasteurization isolates and 3 (10%) were raw milk isolates. Automated ribotyping revealed that raw and pasteurized Pseudomonas fluorescens isolates were indistinguishable (similarity index > 0.93), suggesting the possibility of postpasteurization contamination with bacteria from raw product. In the plant, filler nozzles were identified as the primary reservoirs of postpasteurization contamination. Nozzle replacement produced significantly lower finished-product PBCs at 7 days postprocessing (> 4-log reduction) and extended fluid product shelf life.

Animals↗

PubMed1997

Assessment of microsatellite alterations in young patients with gastric adenocarcinoma.

BACKGROUND: Genetic factors are probably important in the development of gastric carcinoma in young patients (younger than 40 years). The authors investigated early onset primary gastric adenocarcinomas for the presence of microsatellite instability, which is a phenotypic marker for the hereditary nonpolyposis colon carcinoma syndrome. METHODS: DNA was extracted from archival microdissected carcinoma and corresponding normal tissue from 10 British gastric carcinoma patients age 19 to 39 years at the time of diagnosis. A panel of 12 microsatellite loci were amplified by fluorescent polymerase chain reaction and analyzed using an automated DNA sequencer. RESULTS: There was no evidence of microsatellite instability. In contrast, allelic imbalance was recorded at D3S966, D3S1076, D10S197, D11S904, P53, NM23, and DCC microsatellite loci. CONCLUSIONS: The authors reported ten cases of early onset gastric carcinoma that demonstrated allelic imbalance but no evidence of instability at microsatellite loci. It is unlikely that defective DNA mismatch repair is important in this group of young patients.

Adenocarcinoma↗

PubMed2004

What SNP genotyping errors are most costly for genetic association studies?

Which genotype misclassification errors are most costly, in terms of increased sample size necessary (SSN) to maintain constant asymptotic power and significance level, when performing case/control studies of genetic association? We answer this question for single-nucleotide polymorphisms (SNPs), using the 2x3 chi(2) test of independence. Our strategy is to expand the noncentrality parameter of the asymptotic distribution of the chi(2) test under a specified alternative hypothesis to approximate SSN, using a linear Taylor series in the error parameters. We consider two scenarios: the first assumes Hardy-Weinberg equilibrium (HWE) for the true genotypes in both cases and controls, and the second assumes HWE only in controls. The Taylor series approximation has a relative error of less than 1% when each error rate is less than 2%. The most costly error is recording the more common homozygote as the less common homozygote, with indefinitely increasing cost coefficient as minor SNP allele frequencies approach 0 in both scenarios. The cost of misclassifying the more common homozygote to the heterozygote also becomes indefinitely large as the minor SNP allele frequency goes to 0 under both scenarios. For the violation of HWE modeled here, the cost of misclassifying a heterozygote to the less common homozygote becomes large, although bounded. Therefore, the use of SNPs with a small minor allele frequency requires careful attention to the frequency of genotyping errors to ensure that power specifications are met. Furthermore, the design of automated genotyping should minimize those errors whose cost coefficients can become indefinitely large.

Alleles↗

PubMed2005

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii.

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry. Proteins were extracted from the bacterial cells, separated by reversed-phase liquid chromatography and mass analyzed. At the end of the chromatography run, the uncharged masses of the multiply charged proteins were determined via automated software routines. The resulting data provided an accurate mass expression profile of the proteins found in the individual strains. From the individual expression profiles, it was possible to identify unique proteins corresponding to strains with thermal resistance. One protein found only in the thermal tolerant strains was sequenced and identified as homologous to a hypothetical protein found in the thermal tolerant bacteria, Methylobacillus flagellatus KT. The protein sequence of this protein was then used to reverse-engineer PCR primers for the gene sequence associated with the protein. In all cases, only thermal tolerant strains of E. sakazakii produced amplified PCR products, demonstrating the specificity of this biomarker.

Amino Acid Sequence↗

PubMed1999

Novel and recurrent mutations in keratin 10 causing bullous congenital ichthyosiform erythroderma.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited keratinizing disorder characterized by erythroderma and blistering in neonates and generalized epidermolytic hyperkeratosis (EH) in adulthood. Previously, it has been shown that BCIE can be caused by mutations in either of the genes encoding K1 or K10, the keratins predominantly expressed in suprabasal layers of the epidermis. Using direct sequencing of genomic PCR fragments, we have analyzed 4 British families with BCIE, all of whom were found to carry mutations in K10. In 1 family, the affected person was found to have an unusual dinucleotide transversion mutation, 2138CC-->AA, causing two amino acid substitutions, D155E and R156S, also in the 1A domain of the K10 polypeptide. In 2 further kindreds, the previously reported "hotspot" mutations 2139C-->T and 2140G-->A were found. These mutations predict amino acid substitutions in the helix 1A domain of K10, designated R156C and R156H respectively. The proband in the fourth family was found to carry a novel mutation 4724T-->C, predicting the amino acid change L452P in the helix 2B domain of K10. All mutations were confirmed in the affected persons and were excluded from a population of 50 normal, unrelated individuals by restriction enzyme analysis. The location of these mutations in the highly conserved helix boundary motif sequences of K10 are consistent with previously reported dominant negative mutations in K10 and other keratins. Despite the unusual nature of two of these mutations, in particular the double missense mutation, the phenotypes of the affected individuals in these 4 families were entirely typical of BCIE.

Amino Acid Substitution↗

PubMed1996

Biochemical phenotypes of Escherichia coli in dogs: comparison of isolates isolated from bitches suffering from pyometra and urinary tract infection with isolates from faeces of healthy dogs.

Escherichia coli from dogs suffering from pyometra and/or urinary tract infection (UTI) were compared with faecal E. coli. We studied 79 isolates from pyometra, 96 from UTI, 91 from faeces of healthy dogs and 60 from faeces of bitches with pyometra. The isolates included 16 bitches with pyometra where E. coli were isolated from both the uterus and the urinary tracts. All isolates were typed with the aid of an automated typing system for biochemical fingerprinting (the PhP-EC system). The PhP types of uterine E. coli isolates from bitches with pyometra showed higher homogeneity, calculated as the mean of similarity between isolates (rmean = 0.79) than epidemiologically unrelated isolates, such as those isolated from faeces (rmean = 0.69) or UTI (rmean = 0.70). This finding suggests that many of the pyometra isolates derives from related clones. Mucoid and nonmucoid uterine isolates from the same sample were generally of the same PhP type. In all 10 cases uterine E. coli isolates were identical or very similar to isolates from faeces of the same bitch. Furthermore, E. coli isolates from both UTI and the infected uterus in the same bitch were identical or very similar in 88% of the cases. It was concluded that E. coli associated with canine pyometra derive from the faecal flora and that the urinary tract is infected by the same E. coli clone as the uterus of a bitch with pyometra.

Animals↗

PubMed2004

Evaluation of the BD PHOENIX automated microbiology system for detection of methicillin resistance in coagulase-negative staphylococci.

The new BD PHOENIX automated microbiology system (Becton Dickinson Diagnostic Systems, Sparks, Md.) is designed for automated rapid antimicrobial susceptibility testing and identification of clinically relevant bacteria. In our study, the accuracy and speed of the BD PHOENIX oxacillin MIC determination for detecting methicillin resistance was evaluated for 200 clinical isolates of coagulase-negative staphylococci (CoNS). Compared to mecA PCR, the BD PHOENIX system detected methicillin resistance with a sensitivity of 99.2%. According to the actual NCCLS oxacillin MIC breakpoint of > or =0.5 microg/ml, the specificity was only 64.9%, attributable to false-positive results in 26 mecA-negative strains, including 16 non-Staphylococcus epidermidis strains. Alternative oxacillin breakpoints of > or =1, > or =2, and > or =4 microg/ml resulted in increased specificities of 83.8, 94.6, and 100% and high sensitivities of 99.2, 99.2, and 96.7%, respectively. Similarly, NCCLS broth microdilution oxacillin MICs exhibited a sensitivity of 100% but a low degree of specificity. However, the previous oxacillin MIC breakpoint of > or =4 microg/ml performed with a sensitivity of 98.4% and a specificity of 98.7%. BD PHOENIX oxacillin MIC results were available after 9 h for 40.5% of the examined CoNS strains and were completed after 17 h. Our results revealed the high reliability of the BD PHOENIX system as a phenotypic method for detection of resistance to oxacillin in mecA-positive CoNS. However, for the improvement of specificity, reevaluation of the optimal oxacillin MIC breakpoint for CoNS appears to be necessary.

Anti-Bacterial Agents↗

PubMed2001

Evaluating phenotype and genotype of drug-resistant strains in herpesviruses.

The isolation of drug-resistant strains of herpesviruses, including Herpes Simplex Virus type I (HSV-1) and type 2 (HSV-2), Varicella-Zoster Virus (VZV), and cytomegalovirus (CMV), has been reported with increasing frequency in immunocompromised patients and is a matter of major concern. Determination of antiviral drug susceptibilities is a prerequisite for the management of drug-resistant herpesvirus infections. Phenotyping studies should be correlated with genotyping, i.e., characterization of the mutations in the target genes. The isolation of drug-resistant virus in the laboratory and the determination of their phenotype and genotype may be useful to clarify the mechanisms of selective drug action. We describe here the procedures used for in vitro selection of drug-resistant herpesvirus mutants and the determination of their patterns of drug-susceptibility. The subcloning of the HSV-1 DNA polymerase gene is described as an example of the methodology followed to determine the mutation(s) in the drug-target viral gene that are associated with the resistant phenotype. To avoid the introduction of mutations by PCR amplification, all subcloning experiments were executed directly on viral DNA. Viral DNA was prepared from each plaque-purified viral strain and a 3.4 kb BamHI fragment containing 87% of the HSV-1 DNA polymerase gene coding region was purified and further digested with SacI; the two resulting fragments were subcloned into pU18 and propagated in Escherichia coli. Plasmid DNA was isolated and the inserts were sequenced using dideoxynucleotide chain termination method with T7 DNA polymerase and Taq DNA polymerase in an automated laser fluorescent DNA sequencer. pUC/M13 reverse, universal primers and oligonucleodite primers based on the wild-type virus sequence were used. The nucleotide sequences of the DNA polymerase genes of the different mutants was then compared with the nucleotide sequence of the wild-type HSV-1 KOS strain.

Animals↗

PubMed2002

First genomic localization of oculo-oto-dental syndrome with linkage to chromosome 20q13.1.

PURPOSE: To characterize the phenotype of autosomal dominant oculo-oto-dental (OOD) syndrome, map the disease locus in a five-generation British family, and evaluate a candidate gene. METHODS: Full clinical assessments in all affected patients included slit lamp and retina examination, refraction, A-scan ultrasound, audiograms, and dental assessments. Genomic DNA from all family members was genotyped, by polymerase chain reaction, for polymorphic genetic markers covering the entire genome. Two-point LOD scores were generated using a linkage analysis suite of computer programs. The gene for eyes absent 2 (EYA2) was screened for mutations by direct automated sequencing and Southern blot analysis. RESULTS: All the affected individuals examined had iris and retina coloboma associated with high-frequency, progressive, sensorineural deafness and globodontia. This is the only genetic disease known to result in pathologically enlarged teeth. The locus for OOD (OOD1) was mapped to 20q13.1. A maximum two-point LOD score of 3.31 was obtained with marker locus D20S836 at a recombination fraction of theta; = 0.00. Two critical recombinations in the pedigree positioned this locus to a region flanked by marker loci D20S108 and D20S159, giving a critical disease interval of 12 centimorgans (cM). Mutation screening of one candidate gene, EYA2, revealed no disease-associated mutations or polymorphic variants. CONCLUSIONS: This is the first genetic localization for the OOD phenotype (ODD1). The disease-causing gene is localized within a 12-cM critical region of chromosome 20q13.1. The identification of the disease gene is not only relevant to the study of vision and hearing defects, but also highlights an exceptional gene involved in the development of human dentition.

Adult↗

PubMed1990

Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone.

Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.

Animals↗

PubMed2001

On reporting fold differences.

As we enter an age in which genomics and bioinformatics make possible the discovery of new knowledge about the biological characteristics of an organism, it is critical that we attempt to report newly discovered "significant" phenotypes only when they are actually of significance. With the relative youth of genome-scale gene expression technologies, how to make such distinctions has yet to be better defined. We present a "mask technology" by which to filter out those levels of gene expression that fall within the noise of the experimental techniques being employed. Conversely, our technique can lend validation to significant fold differences in expression level even when the fold value may appear quite small (e.g. 1.3). Given array-organized expression level results from a pair of identical experiments, our ID Mask Tool enables the automated creation of a two-dimensional "region of insignificance" that can then be used with subsequent data analyses. Fundamentally, this should enable researchers to report on findings that are more likely to be in nature truly meaningful. Moreover, this can prevent major investments of time, energy, and biological resources into the pursuit of candidate genes that represent false positives.

Computational Biology↗

PubMed2005

Parieto-occipital grey matter abnormalities in children with Williams syndrome.

Williams syndrome (WS) is a neurodevelopmental disorder resulting from a hemizygous deletion of chromosome 7q11.23. The phenotype of WS consists of typical dysmorphic features, supravalvular aortic stenosis, infantile hypercalcemia and growth retardation. While language and facial recognition seem to be relatively spared, visuospatial constructive disabilities are a hallmark of the neurobehavioral profile of WS. In order to search for actual structural abnormalities underlying this precisely defined neurodevelopmental disorder, we performed anatomical magnetic resonance imaging (MRI) in 9 WS children (11.6 +/- 3.1 years; age range: 5.5-15 years) and 11 normal age-matched control children (11.8 +/- 2.2 years; age range: 8-15 years) using voxel-based morphometry (VBM). VBM is a fully automated whole-brain technique that delivers a voxel-wise assessment of regional grey and white matter concentration. A significant decrease in grey matter concentration was detected in the left parieto-occipital region of WS children (P < 0.05 corrected height threshold). The location of this abnormality in WS children coincides with the location of the structural abnormality previously described using the same method in 13 WS adults. These parieto-occipital abnormalities are consistent with the cognitive profile of WS which includes severe visuospatial construction and numerical cognition deficits. The demonstration of identical structural abnormalities in both adults and children argues for their early origin. Additionally, our study provides support for the use of advanced structural imaging techniques in children, in order to improve our understanding of neurobehavioral phenotypes associated with well-defined genetic disorders.

Adolescent↗

PubMed2005

Automated use of mutagenesis data in structure prediction.

In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods.

Algorithms↗