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PubMed2005

Validation of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E.

The strategy for screening of thalassemia and Hb E by a combination of osmotic fragility (OF) test and dichlorophenol indophenol precipitation (DCIP) test was validated with 436 unrelated Thai subjects. Hemoglobin (Hb) typing, Hb A2 quantitation, PCR and DNA sequence analysis were used as confirmatory methods for diagnosis of thalassemia and hemoglobinopathy. The sensitivity and specificity of this strategy was 100% and 79.7%, respectively. The results assessed by two medical scientists were exactly the same with 93.3% accuracy in comparison with the confirmatory methods. A combination of OF and DCIP has been shown to be a reliable, rapid, simple and sensitive strategy for screening thalassemia and Hb E in the Thai population.

2,6-Dichloroindophenol↗
PubMed2003

[Prader-Willi syndrome and genomic imprinting].

OBJECTIVE: Prader-Willi syndrome (PWS) is an example of a human genetic disorder that involves imprinting genes on the proximal long arm of chromosome 15 and SNRPN gene as a candidate gene for this syndrome. The purpose of this study was to show the molecular genetic defects and genomic imprinting basis in Chinese PWS patients and to evaluate the clinical applications of a differential diagnostic test for PWS. METHODS: Fluorescence in situ hybridization (FISH) and methylation-specific PCR (MSPCR) techniques were applied for 4 clinically suspected PWS patients. Using three probes, including SNRPN probe for identification of the critical locus in PWS region, D15Z1 and PML control probes for identification of the 15p arm and 15q arm, the authors detected the deletions 15q in PWS. MSPCR was based on sodium bisulfite treatment of DNA and PCR primers specific for the maternal and paternal allele. RESULTS: When hybridized with mixed probes, it was found in 2 patients that the central specific signal was absent, but both the flanking control signals were retained, indicating SNRPN gene deletion of chromosome 15q11-13. Bisulfite-modified DNA from all PWS children amplified with methylated allele-specific primer pair showed only maternal 131bp PCR product, indicating the maternal uniparental disomy (UPD15). CONCLUSION: Genomic imprinting plays an important role in the molecular pathogenesis of PWS that caused by paternal microdeletions of 15q11-q13 or maternal UPD of chromosome 15. The basic defect seemed to be an absence of function of PWS genes that are normally expressed only from the paternal chromosome 15. MSPCR is a rapid and simple PCR-based assay compared with other cyto-molecular tests and its results were consistent with the clinical diagnosis of PWS, so it seems to be a reliable diagnostic method for PWS patients who show abnormal methylation at SNRPN. The genetic differential tests for PWS are important in determining familial recurrence risk.

Adolescent↗
PubMed1995

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes.

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.

Biopsy↗
PubMed1977

Mutations causing rapid development of Dictyostelium discoideum.

A mutation affecting the speed of slime mold development has been genetically analyzed. Strain FR17 carries a recessive mutation on linkage group IV. A selection procedure for isolating more mutants of this type has been developed and new mutations have been tested for complementation. The aberrant morphology of these strains can be partially corrected by development in the presence of glucose.

Dictyostelium↗
PubMed2005

Increased levels of GFAP in the cerebrospinal fluid in three subtypes of genetically confirmed Alexander disease.

GFAP levels in the CSF were highly elevated in three genetically confirmed cases of Alexander disease clinically conforming with infantile, early and late juvenile forms. No other CSF abnormalities were detected. Assay of CSF-GFAP may prove to be a rapid and cost-effective screening test in clinical variants of Alexander disease and an indicator of GFAP gene mutations.

Adult↗
PubMed2006

Methods to rapidly and accurately screen a large number of ENU mutagenized mice for abnormal motor phenotypes.

In a dominant genetic screen for late-onset motor impairments in mice, 16-20-week-old N-nitroso-N-ethylurea (ENU)-mutagenized females were subjected to a behavioural test battery consisting of a visual assessment followed by the vertical pole, rotarod and grip strength tests. SOD1-G93A transgenic mice were tested in parallel as a positive control to provide information on the validity and sensitivity of the screen. From among the 1500 G1 ENU mice screened, four affected mice with impaired motor function were classified as outliers. Approximately 32% of the G2 and G3 progeny of one outlier were affected. Vertical pole, rotarod and grip strength test scores were significantly correlated with each other and with body weight in the G1 progeny, but the correlation with body weight was not maintained in the G2 and G3 progeny. We found that two tests, tail suspension and vertical pole, were sufficient to distinguish ENU outliers and SOD1-G93A hemizygotes from control mice, and could detect abnormalities earlier and more frequently than the other tests employed.

Animals↗
PubMed1974

Genetic analysis of an Escherichia coli mutant with a lesion in stable RNA turnover.

A mutant that rapidly degrades more than 80% of its rRNA and tRNA under defined conditions was genetically analyzed. Two genes, srnA and srnB, are separately located, and the mutated alleles of both are required for degradation of stable RNA in cultures treated with rifampicin at 42 degrees . srnA is closely linked to tsx by matings and transduction tests; by P1 transduction, the gene order is lac (9 min) proC (9.55 min) tsx (9.8 min) srnA (about 10 min) purE (12 min) rnsA (14.4 min). srnB is not yet completely mapped, but is outside the lac-rnsA region, probably in the region between 75 and 90 min.-The product of the rnsA gene, RNase I, is a potent endonuclease of E. coli, and the only one known that can attack ribosomes and tRNA. However, not only are the srn lesions genetically separate from rnsA, but also, derivatives of an srn strain were prepared lacking RNase I, and they retain the Srn(-) phenotype. Thus, no correlation of rapid RNA turnover and RNase I activity has been found.

Alleles↗
PubMed1998

The fundamental and medical impacts of recent progress in research on hereditary hearing loss.

What would define real progress in the field of deafness research in fundamental and medical terms? In fundamental terms, progress would be measured by an improvement in our knowledge of the development and physiology of the ear. In medical terms, progress would lead to the division of the broad category of hearing defects into distinct clinical entities or subclasses, the collection of epidemiological data, the creation of molecular diagnostic tests, the improvement of genetic counselling services and the development of new therapeutics. In this review, we will introduce some general considerations on hereditary hearing loss and on the structure and function of the ear, present the rapidly emerging data on the molecular basis of syndromic and non-syndromic forms of hearing loss and comment on relevant recent progress in this field of research. Generally speaking, the isolation of genes underlying hereditary hearing loss has, as yet, had little impact on our understanding of the biology of the ear, whereas it has made major contributions to the medical field, in particular due to the recognition of two genes, Cx26 and mitochondrial 12S rRNA , as frequently underlying cases of non-syndromic hearing impairment.

Animals↗
PubMed2004

A toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system.

Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Some cationic amphiphilic compounds are known to have the potential to induce phospholipidosis. This study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. In electron microscopy, HepG2 cells developed lamellar myelin-like bodies in their lysosomes, the characteristic change of phospholipidosis, after treatment with these compounds for 72 h. DNA microarray analysis performed 6 and 24 h after treatment showed alterations in gene expression reflecting the inhibition of lysosomal phospholipase activity and lysosomal enzyme transport, and the induction of phospholipid and cholesterol biosynthesis. Seventeen genes that showed a similar expression profile following treatment were selected as candidate markers. Real-time PCR analysis confirmed that 12 gene markers showed significant concordance with lamellar myelin-like body formation. Furthermore, the average fold change values of these markers correlated well with the magnitude of this pathological change. In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.

Carcinoma, Hepatocellular↗
PubMed2001

Pharmacogenetics: the Dx perspective.

The promise of the rapidly developing field of pharmacogenetics is that genetically determined propensities of individual patients to respond favorably or adversely to a given pharmacologic agent will be able to be determined prior to administration of that drug. The realization of that promise, however, is predicated on a number of developments in the capabilities of diagnostic laboratories. These developments include the introduction of automated technologies for efficiently and accurately detecting, quantifying and decoding specific nucleic acid sequences and the concomitant availability of information technology-based applications for rapidly analyzing, interpreting and then communicating complex genetic data to healthcare providers. This article will review currently available and developmental molecular diagnostic technology and in addition, describe the current status and speculate on the future of pharmacogenetic testing in the clinical laboratory.

Humans↗
PubMed2003

Detection of the most frequent mutations in BRCA1 gene on polyacrylamide gels containing Spreadex Polymer NAB.

Heterozygous carriers of germ-line mutations in the BRCA1 gene are at high risk for the development of breast and ovarian cancer. Inactivating mutations have been identified in the whole coding region of the gene, however, repeatedly occuring mutations can explain a large proportion of gene alterations detected in certain ethnic groups. In Czech patients, the 5382insC and 185delAG mutations may account for approximately 50% of all BRCA1 abnormalities (unpublished data). In the present study, a rapid and simple method to identify these short insertions and deletions that alter the size of the polymerase chain reaction (PCR) product is described. The analysis involves the separation of fragments amplified with primers that flank altered sites in the BRCA1 gene on non-denaturing polyacrylamide gels containing Spreadex Polymer NAB. The increased resolving power of Spreadex gels enables full separation of two DNA fragments that differ by 1-bp on gels that are 5 cm long. The method gave interpretable results with the genetic material obtained from all tested mutation carriers and control persons. Defective alleles were also detected in DNA samples from carriers of the 1135delA mutation in BRCA1 and the 4206ins4 mutation in BRCA2. These results suggest that electrophoresis on Spreadex gels can be used universally for detection of the most frequent frameshift mutations in BRCA genes. The method is suitable even for rapid screening of frequent germ-line mutations in BRCA genes in breast and ovarian cancer patients not selected for family history of cancer or age at diagnosis.

Acrylic Resins↗
PubMed2005

Tissue safety in view of CJD and variant CJD.

Epidemiological studies on human transmissible spongiform encephalopathies (Creutzfeldt-Jakob Disease, CJD) have shown that the agent could be transmitted by highly infectious tissues like brain, spinal cord or retina and medicinal products derived from these tissues (i.e. human growth hormone, dura mater). A few cases of transmission of CJD by neurosurgical instruments have been reported. The transmission of the agent of variant CJD, which is suspected to be transmitted by BSE-contaminated food, by blood transfusion implies that in contrast to the agent of classical CJD this agent can also be transmitted by organs and tissues other than nerve tissues. Health authorities have implemented guidelines to reduce the risk of transmission of human and animal TSE by human and veterinary medicinal products. The high resistance of TSE agents against physical or chemical treatment hamper the development of highly efficient inactivation steps in the production of medicinal products. Donor selection is considered as an efficient measure to reduce the risk of TSE transmission. However, the development of rapid, sensitive and specific diagnostic test systems is urgently required to test blood, organs and tissue of donors.

Animals↗
PubMed2001

Use of a genetic algorithm for the analysis of eye movements from the linear vestibulo-ocular reflex.

It is common in vestibular and oculomotor testing to use a single-frequency (sine) or combination of frequencies [sum-of-sines (SOS)] stimulus for head or target motion. The resulting eye movements typically contain a smooth tracking component, which follows the stimulus, in which are interspersed rapid eye movements (saccades or fast phases). The parameters of the smooth tracking--the amplitude and phase of each component frequency--are of interest; many methods have been devised that attempt to identify and remove the fast eye movements from the smooth. We describe a new approach to this problem, tailored to both single-frequency and sum-of-sines stimulation of the human linear vestibulo-ocular reflex. An approximate derivative is used to identify fast movements, which are then omitted from further analysis. The remaining points form a series of smooth tracking segments. A genetic algorithm is used to fit these segments together to form a smooth (but disconnected) wave form, by iteratively removing biases due to the missing fast phases. A genetic algorithm is an iterative optimization procedure; it provides a basis for extending this approach to more complex stimulus-response situations. In the SOS case, the genetic algorithm estimates the amplitude and phase values of the component frequencies as well as removing biases.

Algorithms↗
PubMed2004

Impact of founder population, drift and selection on the genetic diversity of a recently translocated tree population.

Recently established, temperate tree populations combine a high level of differentiation for adaptive traits, suggesting rapid genetic evolution, with a high level of genetic diversity within population, suggesting a limited impact of genetic drift and purifying selection. To study experimentally the evolutionary forces in a recently established population, we assessed the spatial and temporal patterns of genetic diversity within a disjunct population of Cedrus atlantica established 140 years ago in south-eastern France from a North African source. The population is expanding through natural regeneration. Three generations were sampled, including founder trees. We analysed 12 isozyme loci, three of which were previously found in tight association with selected genes, and quantitative traits. No bottleneck effect was detected in the founder generation, but a simple test of allelic association revealed an initial disequilibrium which disappeared in the following generations. The impact of genetic drift during secondary evolution was limited, as suggested by the weak temporal differentiation. The genetic load was not reduced after 3 generations, and the quantitative variation for adaptive traits did not change either. Thus, initial genetic changes first proceed from a rapid re-organisation of the diversity through mating and recombination, whereas genetic erosion through drift and selection is delayed due to temporal and spatial stochasticity. Two life-history traits of trees contribute to slowing down the processes of genetic erosion: perenniality and large spatial scale. Thus, one would expect recently established tree populations to have a higher diversity than older ones, which seems in accordance with experimental surveys.

Biological Evolution↗
PubMed2003

Rapid detection of six common Mediterranean and three non-Mediterranean alpha-thalassemia point mutations by reverse dot blot analysis.

We describe the implementation of reverse dot blot (RDB) hybridization as a rapid nonradioactive method for the identification of six frequent globin gene point mutations in the Mediterranean population: alpha(Hph)alpha: alpha2 IVS I donor site GGTGAGG --> GG-----; alpha(NcoI)alpha: alpha2 initiation codon ATG --> ACG; alpha(TSaudi)alpha: alpha2Poly A signal AATAA --> AATAAG; alpha(Icaria)alpha: alpha2 termination codon TAA --> AAA (Ter --> LYS); alpha(CS)alpha: alpha2 termination codon TAA --> CAA (Ter --> gly); alphaalpha(NcoI): alpha1 initiation codon ATG --> GTG; and three alpha2 globin gene point mutations found in immigrants in Italy: alpha(T-Quongsze)alpha: alpha2 codon 12 CTG --> CCG (Leu --> Pro); alpha(Seal Rock)alpha: alpha2 termination codon TAA --> GAA (TER --> GLU); and alpha(Koyadora)alpha: alpha2 termination codon TAA --> TCA (TER --> SER). The method uses the principle of allele-specific oligonucleotide (ASO) hybridization, but it is a nonradioactive method and permits rapid and simultaneous typing of point mutations and small deletions.

Alleles↗
PubMed1997

Rapid mutation screening in type 2A von Willebrand's disease using universal heteroduplex generators.

Patients with type 2A von Willebrand's disease (VWD) commonly have missense mutations in the A2 domain of the von Willebrand factor (VWF) protein. This domain is encoded by the 3' region of VWF gene exon 2 8 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA-based diagnostic technique which enables at least 10 previously described mutations to be rapidly identified. The method involves polymerase chain reaction (PCR) amplification of two exon 28 gene segments between codons 717-788 and 803-893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletions contiguous to the sites of known mutations and, following hybridization, allele-specific heteroduplexes are generated which can be detected by simple polyacrylamide gel electrophoresis and ethidium bromide staining. A small panel of UHG molecules covering the 3' region of exon 28 should enable the large majority of type 2A VWD patients to be rapidly diagnosed by genotype.

Base Sequence↗
PubMed1992

Production of active Serratia marcescens metalloprotease from Escherichia coli by alpha-hemolysin HlyB and HlyD.

Serratia marcescens produces an abundant extracellular metalloprotease. The gene for this protease had previously been cloned and expressed in Escherichia coli, in which no functional protease could be found. However, the protease gene carries the LXGGXGND repeat motif found in alpha-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the alpha-hemolysin hlyB and hlyD transport determinants are sufficient to allow secretion and activation of a functional metalloprotease species from E. coli, as are the comparable protease secretion functions of Erwinia chrysanthemi. However, strains expressing protease with the hlyBD transport system are unstable and rapidly lose the ability to produce functional protease.

DNA, Bacterial↗
PubMed1995

MudSacI, a transposon with strong selectable and counterselectable markers: use for rapid mapping of chromosomal mutations in Salmonella typhimurium.

The transposable bacteriophage Mu and its mini-Mu derivatives are useful tools for the genetic analysis of many bacteria. A variety of antibiotic-resistant Mu derivatives have been constructed, allowing direct selection for cells which contain the transposon. However, in many cases a counterselection against the transposon would greatly facilitate further genetic analysis. In this paper we report the construction of MudSacI, a mini-Mu derived transposon containing the sacB (secretory levansucrase) gene of Bacillus subtilis, which confers sucrose sensitivity upon gram-negative bacteria. We describe the use of this transposon as a tool for rapid genetic mapping of chromosomal genes in Salmonella typhimurium. Simple modifications of this approach should facilitate rapid mapping in many other bacteria as well.

Bacteriophage mu↗