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PubMed2004

RpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis strains isolated in the Aegean region of Turkey.

Although DNA sequencing method has not been used routinely for detecting resistance of Mycobacterium tuberculosis to antituberculosis drugs, it is suggested for the investigation of gene mutations causing resistance. Using DNA sequencing (Automated Applied Biosystems), we attempted to determine mutations in the 81-bp cor region (rifampin resistance determining region) of the rpoB gene in 48 Mycobacterium tuberculosis strains found to be rifampin resistant by classical phenotypic methods. Of the 48 strains, 46 (95.8%) were found to have rpo gene mutations with 13 different types while in two (4.2%) of the 48 strains, no mutations were detected. None of the strains had mutations at more than one codon. Point mutations at the 531st (52.1%) and 526th (18.9%) codons were frequent. The most frequent point mutation was Ser531Leu, and it was found in 21 (43,8 %) of 48 strains. This is the first study from Turkey, reporting Ser522Leu point mutation in one isolate and deletion of 515th codon (ATG - Met) in two isolates.

Antitubercular Agents↗

PubMed1993

Viral markers in HIV infection and AIDS.

Viral and immune markers are used for monitoring either progression of human immunodeficiency virus (HIV) disease or response to antiviral therapy. Ideal properties of viral markers are that they are present in all HIV-infected persons at all stages of disease, that they are related to disease pathogenesis, that they can be easily quantitated, that this quantitation correlates rapidly and predictably with both disease stage and response to antivirals, and that they can be developed into rapid, reproducible automated tests. Currently available viral markers include HIV p24 antigenemia (after acid glycine dissociation), anti-p24 antibody titres, quantitative DNA and RNA polymerase chain reaction performed on cells and plasma, and HIV isolate phenotype. In Australia, these markers have been studied in acute HIV seroconversion, in neonatal infection, in body fluids other than blood, and in monitoring of response to antiviral drug therapy.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Phenotype of cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma.

AIM: To investigate the phenotypes associated with cytochrome P4501B1 gene (CYP1B1) mutations in Japanese patients with primary congenital glaucoma (PCG). METHODS: 66 Japanese patients with PCG were screened for sequence mutations in the CYP1B1 gene using single strand conformation polymorphism analysis followed by automated DNA sequencing. 11 cases had a CYP1B1 mutation in both alleles (the mutation group) and 21 cases did not have a CYP1B1 mutation (the "no mutation" group). The clinical features, such as age of onset, sex, intraocular pressure, and Descemet's membrane rupture, of the two groups were compared. RESULTS: The clinical symptoms and signs did not differ for the two groups. The mean age at onset was 1.7 months in the mutation group and 3.1 months in the no mutation group, and the male:female ratio was 6:5 in the mutation group and 19:2 in the no mutation group. Both of these differences were statistically significant. CONCLUSIONS: In clinically diagnosed cases of PCG, a subgroup shows a CYP1B1 gene mutation. Age at onset was earlier in PCG patients with CYP1B1 mutations than in patients without mutations. Women were more prevalent among patients with mutations than those without mutations.

Age of Onset↗

PubMed1997

A novel variant of lysosomal acid lipase in cholesteryl ester storage disease associated with mild phenotype and improvement on lovastatin.

Cholesterol ester storage disease (CESD) is a rare congenital disorder of lipid metabolism, with mutation of the lysosomal acid lipase gene, causing chronic liver disease, usually before adolescence. We here describe three adult siblings with CESD diagnosed by light microscopic demonstration of excessive lysosomal storage of lipids with accumulation of foamy cells in liver biopsies and by a decrease in acid lipase activity (2-3% of controls). One patient (male, 46a) had extensive liver fibrosis, another (female, 58a) had cirrhosis of the liver. The third patient had died from variceal haemorrhage (female, 56a). Using sequence analysis of RT-PCR products of LAL mRNA, the patients were identified as compound heterozygotes for a G-->A substitution at position -1 of the exon 8 splice donor site and a point mutation at the second allele, resulting in a His108-->Pro shift. In two patients, therapy with lovastatin was initiated, which led to normalisation of serum cholesterol and triglyceride levels. After 12 months, liver biopsy demonstrated a significant decrease in vacuolisation of hepatocytes, with fewer and smaller droplets. Semi-automated computer-assisted image analysis of electron microscopic sections demonstrated a decrease in the hepatocellular lysosomal area from 20.5+/-7.1% to 11.7+/-6.5% (p<0.05) and 41.7+/-5.1% to 33.4+/-4.4% (p<0.01). We conclude that in two siblings with a novel LAL variant and mild phenotype of CESD, lovastatin decreased both serum lipid concentrations and hepatocellular lysosomal content.

Anticholesteremic Agents↗

PubMed2000

Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle.

Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods.

Animals↗

PubMed2003

Persistent oxidative stress in chromosomally unstable cells.

Past work using the human-hamster hybrid line GM10115 has demonstrated that exposure to a variety of DNA damaging agents can lead to the persistent destabilization of chromosomes. To gain insight into the potential biochemical mechanisms involved in perpetuating the unstable phenotype, groups of clones characterized as stable or unstable were analyzed for indications of oxidative stress. All of the clones were derived from single progenitor cells surviving exposure to ionizing radiation or chemicals. Compared with their stable counterparts, unstable clones possessed elevated levels of reactive oxygen species (ROS) as measured by their enhanced ability to oxidize fluorogenic dyes. Fluorescence automated cell sorting analysis indicated that unstable clones had significantly higher mean fluorescence signals of approximately 2-fold and approximately 1.25-fold, respectively, as derived from the dyes 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, respectively. To determine whether mitochondria might constitute a potential source of ROS, stable and unstable clones of cells were analyzed for mitochondrial content using nonyl acridine orange and function using rhodamine 123. Fluorescence automated cell sorting data indicated that compared with stable clones, unstable clones possessed an elevated number (15% increase in mean nonyl acridine orange fluorescence) of dysfunctional mitochondria (27% decrease in mean rhodamine 123 fluorescence). Interestingly, the consequences of elevated ROS did not translate to an increase in oxidative base damage in nuclear DNA. Analysis of nine different base damage adducts by gas chromatography/mass spectrometry did not reveal significant differences between stable and unstable clones. The data suggest that the perpetuation of many of the abnormal phenotypes associated with genomic instability may be linked to a state of chronic oxidative stress derived in part from dysfunctional mitochondria.

Aneuploidy↗

PubMed2002

Intravenous cocaine and heroin self-administration in rats selectively bred for differential saccharin intake: phenotype and sex differences.

RATIONALE: Rats selectively bred for high intake of a sweet saccharin solution (HiS) consume more ethanol than their low-saccharin intake (LoS) counterparts. The HiS phenotype may be a predictor of abuse of other drugs via other routes of administration. OBJECTIVE: HiS and LoS, male and female rats were tested for acquisition of IV cocaine and heroin self-administration under a fixed-ratio 1 (FR1) schedule, and cocaine-reinforced behavior was examined under a progressive-ratio (PR) schedule. METHODS: Four groups of rats (HiS males and females and LoS males and females) were trained to self-administer IV cocaine (0.2 mg/kg), and another four groups were trained to self-administer heroin (0.015 mg/kg) using an automated autoshaping procedure. Rats were allowed 30 days to reach a criterion whereby a mean of 100 (cocaine) or 20 (heroin) infusions were self-administered during 6-h sessions over 5 consecutive days. RESULTS: The HiS female rats acquired cocaine self-administration significantly more rapidly than the LoS rats, and females of both phenotypes met the acquisition criteria more rapidly than males. In both HiS and LoS cocaine groups a greater percentage of females (compared with males) met the acquisition criteria within 30 days. The only cocaine group in which 100% met the criterion was the HiS females. The female (compared with male) heroin groups showed a more rapid rate of acquisition, but there was no difference due to saccharin phenotype. In each of the four heroin groups 100% of all rats met the criteria within 30 days. Results of the PR schedule in the HiS females and males and LoS females indicated significantly higher break points in the HiS females (compared with HiS males), but there were no differences in females due to phenotype. CONCLUSION: Female rats selectively bred for higher saccharin intake show more rapid and successful acquisition of IV self-administration of a low dose of cocaine than those bred for low saccharin intake. Female rats (compared with males) consistently showed accelerated rates of acquisition and maintenance (PR) of cocaine self-administration and acquisition of heroin self-administration.

Analysis of Variance↗

PubMed2005

Analysis of the effects of turning bias on chemotaxis in C. elegans.

C. elegans advances up a chemical gradient by modulating the probability of occasional large, course-correcting turns called pirouettes. However, it remains uncertain whether C. elegans also uses other behavioral strategies for chemotaxis. Previous observations of the unusual, spiral-shaped chemotaxis tracks made by the bent-head mutant unc-23 point to a different strategy in which the animal continuously makes more subtle course corrections. In the present study we have combined automated tracking of individual animals with computer modeling to test the hypothesis that the pirouette strategy is sufficient on its own to account for the spiral tracks. Tracking experiments showed that the bent-head phenotype causes a strong turning bias and disrupts pirouette execution but does not disrupt pirouette initiation. A computer simulation of disrupted pirouette behavior and turning bias reproduced the spiral tracks of unc-23 chemotaxis behavior, showing that the pirouette strategy is sufficient to account for the mutant phenotype. In addition, the simulation reproduced higher order features of the behavior such as the relationship between the handedness of the spiral and the side to which the head was bent. Our results suggest that the pirouette mechanism is sufficient to account for a diverse range of chemotaxis trajectories.

Animals↗

PubMed2003

Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.

Ascomycota↗

PubMedDate not supplied

Molecular subtyping methods for Listeria monocytogenes.

Conventional, phenotypic, and DNA-based subtyping methods allow differentiation of Listeria monocytogenes beyond the species and subspecies level. Bacterial subtyping methods not only improve our ability to detect and track human listeriosis outbreaks, but also provide tools to track sources of L. monocytogenes contamination throughout the food system. The use of subtyping methods also provides an opportunity to better understand the population genetics, epidemiology, and ecology of L. monocytogenes. The last 5 years have seen tremendous advancements in the development of sensitive, rapid, automated, and increasingly easy-to-use molecular subtyping methods for L. monocytogenes. This review highlights key aspects of different L. monocytogenes subtyping methods and provides examples of their application in public health, food safety, population genetics, and epidemiology. A significant focus is on the application of subtyping methods to define L. monocytogenes subtypes and clonal groups, which may differ in phenotypic characteristics and pathogenic potential.

Bacterial Typing Techniques↗

PubMed1996

French Caucasian population data obtained from fluorescently detected HUMvWFA31/A and HUMF13A01 short tandem repeat loci.

Allele and phenotype frequencies for two tetranucleotide STR (short tandem repeat or microsatellite) systems, HUMvWFA31/A and HUMF13A01, were obtained from a sample of approximately 240 unrelated individuals randomly selected from the French Caucasian population. PCR (polymerase chain reaction) products were analysed on 6% polyacrylamide denaturing gels and visualized using fluorescently labelled primers on the automated 373A ABI DNA sequencer (Applied Biosystems Inc.). French Caucasian allele frequencies were compared to other published Caucasian data. Conditions were optimised for the quadruplex PCR amplification of these two STR loci together with the HUMFESFPS and HUMTH01 loci and the quadruplex PCR was also performed on various forensic DNA samples.

Alleles↗

PubMedDate not supplied

Platelet transfusion: automated selection of donors.

The selection of platelet donors, according to HLA antigens, has been computerized using a method based on the calculation of incompatibilities and risk of immunization between donors and recipients. Since the risk that an antigen present in a donor will immunize an incompatible recipient of known HLA phenotype, an incompatibility score is established and a donor file printed out, ranked according to the degree of incompatibility. The principle of this method is valuable for any other compatibility system and can be easily extended according to the computer potency. The method allows a rapid selection of the most appropriate plateletpheresis donors to avoid immunizing unsensitized patients or to achieve the best degree of compatibility in previously sensitized patients.

Blood Donors↗

PubMed2002

A high-throughput screening procedure for identifying mice with aberrant taste and oromotor function.

Little is known about how specific genes influence taste function in mammals. One of the most promising ways to fill this void is to screen the progeny of chemically mutagenized (or genetically altered) mice for aberrant taste phenotypes and then identify the mutated gene(s) that is associated with each taste anomaly. To exploit this approach, a high-throughput and robust screening procedure is needed. We have attempted to meet this demand by developing an automated procedure that assesses taste responsiveness of individual mice to palatable and unpalatable taste stimuli. We focused on three taste stimuli (quinine hydrochloride, QHCl; sodium chloride, NaCl; and sucrose) and one mouse strain (C57BL/6). We used a commercially available gustometer system that both monitors the licking responses of mice and controls the presentation of each taste stimulus during successive 5 s trials. We describe a screening procedure that (after 2 days of simple training) can generate a concentration-response curve for NaCl or sucrose during a single 30 min test session, and for QHCl over three 30 min test sessions. A normative database based on the responses of 98 mice subjected to our screening procedure is also presented. We envision that investigators could use this normative database to assess taste function in the progeny of mutagenized (or genetically altered) mice. Any mouse that deviates significantly-e.g. three standard deviations (SD)-from the mean of the normative database would be flagged as having a potentially interesting mutation. We also developed an additional second screen for identifying mice with oromotor abnormalities. This latter screen is necessary because oromotor problems could lead to false positives or negatives in the screen for taste function, but is also useful for researchers interested in genes influencing oromotor circuitry. Throughout the development of the screening protocol, we sought to balance two conflicting demands: the need to maximize the screen's sensitivity and minimize its duration. This screen represents a significant improvement over the common two-bottle preference test because it assesses taste function more specifically and in a fraction of the time.

Animals↗

PubMed1995

Variability in absolute lymphocyte counts obtained by automated cell counters.

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)-infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets. We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built-in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples. Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory.

Automation↗

PubMed1997

Comparison of sib-pair and variance-components methods for genomic screening.

The statistical properties of sib-pair and variance-components linkage methods were compared using the nuclear family data from Problem 2. Overall, the power to detect linkage was not high for either method. The variance-components method had better power for detection of linkage, particularly when covariates were included in the model. Type I error rates were similar to nominal error rates for both methods.

Analysis of Variance↗

PubMed2003

Tissue microarray (TMA) applications: implications for molecular medicine.

Modern expression-screening platforms such as complementary DNA (cDNA) arrays allow for high-throughput lead discovery in cancer and other diseases. For evaluation of promising candidate genes, however, in situ analysis of high numbers of clinical tissues samples--for example, by immunohistochemistry or fluorescence in situ hybridisation--is mandatory. Tissue microarray (TMA) technology greatly facilitates such analysis. Minute tissue cores (diameter 0.6 mm) are removed from up to a thousand different conventional paraffin blocks and re-assembled in a single empty paraffin block at predefined positions. Sections of the resulting TMA can be utilised for the range of research applicable to conventional tissue sections. Important advantages of the TMA technology are speed (parallel analysis of up to a thousand tissues), cost efficiency (the same amount of reagents required for a single large-section analysis is sufficient for a thousand samples), and standardisation (the same experimental conditions are applied to all samples). Because of the high numbers of samples usually included in TMAs, they are optimally suited to detect genotype-phenotype associations with high statistical power. Thus, TMA technology will markedly accelerate the transition from basic research to clinical applications.

Automation↗

PubMedDate not supplied

The phenotype of arg555trp mutation in a large Turkish family with corneal granular dystrophy.

PURPOSE: A large Turkish family with 52 members, 26 of whom had Groenouw type 1 corneal granular dystrophy was evaluated by genetic linkage studies and mutation analyses. Phenotype-genotype correlations were also assessed. METHODS: DNA from peripheral blood lymphocytes of 22 family members was used in establishing linkage to chromosome 5q31. Single-strand conformation polymorphism analysis was done to detect mutations in exons 4 and 12 of the human transforming growth factor beta-induced gene located on chromosome 5q31. Automated sequencing was performed on exon 12 of an affected patient. RESULTS: Patients yonger than 15 years of age had typical linear, granular opacities whereas adults had coarser, deeper granular stromal deposits. These changes were not associated with recurrent erosions or significant visual disabilities. The family was linked to chromosome 5q31 and a DNA shift was observed on exon 12 of affected patients. CGG to TGG transition producing R555W mutation was found. CONCLUSIONS: Segregation of Arg555Trp has been described as causing Groenouw type I corneal dystrophy of variable severity in patients of various ethnic backgrounds. In this large Turkish pedigree, the Arg555Trp mutation was associated with a mild phenotype that became clinically evident at five years of age but which remained asymptomatic in terms of corneal erosions.

Adolescent↗

PubMed2000

Evidence for a new locus for X-linked retinitis pigmentosa (RP23).

PURPOSE: X-linked retinitis pigmentosa (XLRP) is a degenerative disease of the retina characterized in the early stages of disease by night blindness as a result of rod photoreceptor loss, progressing to severe disease with loss of central vision by the third decade in affected males. XLRP displays exceptional genetic heterogeneity, with five reported loci on the human X-chromosome. To investigate the level of heterogeneity for XLRP in the patient pool in the current study, extensive haplotype analysis, linkage analysis, and mutation screening were performed. METHODS: Haplotype analysis of a family with diagnosed XLRP was scored with more than 34 polymorphic markers spanning the entire X-chromosome, including regions already identified as harboring XLRP genes and retina-specific genes. Two-point and multipoint lod scores were calculated. Affected male DNA was amplified with primers specific for the retinoschisis gene (XLRS1), and the products were screened for nucleic acid alterations by direct automated sequencing. RESULTS: In this article haplotype and linkage data are presented identifying a new locus for XLRP on the short arm of the X-chromosome, distinct from previously reported gene localizations for XLRP. The phenotype is atypical, in that the onset of vision loss in the male members of this family is unusually early, and female obligate carriers have normal fundi and waveforms. Informative recombination events in this family define a locus for XLRP (RP23) on Xp22 between the markers DXS1223 and DXS7161, spanning approximately 15 cM. A maximum lod score of 2.1 was calculated for the locus order DXS7103-8 cM-(RP23/DXS1224)-4 cM-DXS999. This new locus (RP23) encompasses the retinoschisis disease gene; therefore, XLRS1 was screened for a mutation. No sequence alteration was identified indicating that mutations in the coding region of the gene responsible for retinoschisis do not cause RP23. CONCLUSIONS: The results describe evidence for a new locus for XLRP (RP23), adding to the established genetic heterogeneity for this disease and the number of genes expressed in ocular tissue residing on the X-chromosome.

Adult↗