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PubMed2006

[The development of rapid screening of colorectal cancer by mutation detection in fecal DNA].

Colorectal cancer is one of the leading causes of death in the world. Screening high-risk populations for colorectal cancer can lower both the incidence of and deaths from the disease. Mutation detection in fecal DNA is a new approach of screening patients for colorectal cancer. Compared with colonoscopy and fecal occult-blood test, this approach has the advantages of high specificity, high sensitivity and general acceptability. This paper systematically reviewed genes associated with fecal DNA testing, methods for extracting tumor-specific DNA, and methods for detecting gene mutations. Moreover, the application of mutation detection using fecal DNA for rapid screening of colorectal cancer was briefly discussed.

Colorectal Neoplasms↗
PubMed1999

Genome complexity, robustness and genetic interactions in digital organisms.

Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection. They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study. Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined 'metabolic' rewards. To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness. The complex organisms are more robust than the simple ones with respect to the average effects of single mutations. Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms. Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies. Our findings support the view that interactions are a general feature of genetic systems.

Biological Evolution↗
PubMed2000

Quaternary ammonium block of mutant Na+ channels lacking inactivation: features of a transition-intermediate mechanism.

1. The quaternary ammonium (QA) lidocaine derivative QX-314 (2-(triethylamino)-N-(2,6-dimethylphenyl)-acetamide) induces internal pore blockade of single cardiac Na+ channels enzymatically modified (papain) to eliminate fast inactivation. The mechanism involves dual, interacting blocking modes (rapid and discrete) with binding domains deep in the pore from the cytoplasmic mouth, and where the rapid blocked configuration serves as a transition-intermediate for the development of discrete block. The primary goals of this study were to test for this mechanism in a recombinant Na+ channel genetically engineered to selectively lack fast inactivation, and if present, to explore the underlying structural features. 2. Fast inactivation was removed in rat skeletal muscle mu1 Na+ channels (RSkM1) with an IFM-QQQ mutation in the cytoplasmic III-IV interdomain (QQQ). QQQ was expressed in Xenopus oocytes and single-channel activity was studied in cell-free, inside-out membrane patches. Application of QX-314 (QX, 0-4 mM) to the cytoplasmic membrane surface caused two distinct modalities of single-channel blockade: reduction of unitary current and interruptions of current lasting tens of milliseconds. These are consistent with rapid and discrete pore block, respectively. The voltage and concentration dependence of block indicates that the modes interact and have binding sites that share a deep location in the pore, at approximately 65 % of the membrane electric field in from the cytoplasmic mouth. 3. Mutation of phenylalanine (F1579) in domain IV-S6, critical in local anaesthetic block, to alanine in QQQ (QQQ-F1579A) disabled discrete block but notably failed to alter rapid block, single-channel gating and slope conductance. 4. Amplitude distribution analysis was applied to long bursts (> 50 ms) of QQQ-F1579A activity to investigate the kinetics of rapid block. Computed rapid blocking and unblocking rate constants are 42 000 +/- 18 000 m-1 ms-1 and 82 +/- 22 ms-1, respectively (n = 3, -20 mV). 5. The results support a general transition-intermediate mechanism that governs internal QX and local anaesthetic pore block of voltage-gated Na+ channels and provide insight into underlying structural features.

Algorithms↗
PubMed2002

Rapid detection of the prion protein M129V polymorphism with the LightCycler.

The common single nucleotide polymorphism at codon 129 of the prion protein gene is a key determinant of the genetic susceptibility to Creutzfeldt-Jakob disease (CJD). Recently, a molecular classification of sporadic CJD based on the M129V genotype in conjunction with other determinants was proposed. In the present study, we describe the development and evaluation of a rapid fluorescent-based assay to detect this polymorphism using the LightCycler system. The two polymorphic alleles could be clearly distinguished by their melting points at 52.1 and 60.4 degrees C, representing the 129V and 129M alleles, respectively. These results were confirmed by DNA sequencing. We evaluated our test in 400 patient samples and found no deviations from the expected melting patterns. The calculated allele frequency for the M-allele was 0.66. Thus, we have established a rapid, reliable fluorescent assay for high-throughput detection of the prion protein M129V polymorphism.

Creutzfeldt-Jakob Syndrome↗
PubMed1990

Quantitative genetics of doubled haploid populations and application to the theory of line development.

The line value of a genotype is defined as the expected value of all lines that can be derived from this genotype. Specific genetic effects are defined for this value: only additive and additive by additive epistatic effects are necessary. There is no dominance effect for such a value. A general expression for the covariances between related lines is given. From a design with several lines per haplodiploidized plant taken at random from a population it is possible to estimate the additive variance for line value and the variance of additive by additive epistasis for line value. Variances of higher order epistasis can be estimated with a two-factor mating design in which a cross is replaced by the population of lines that can be derived from it. With a diallel or a factorial design a direct test for the presence of homozygous by homozygous epistasis is possible. The application of the concept of line value to the theory of line development leads to simple expressions of genetic advance in one cycle of recurrent selection according to the testing system. A brief consideration of these expressions leads to the conclusion that single doubled haploid descent recurrent selection will be one of the most efficient methods for low heritabilities and with a rapid development of doubled haploid lines.

Alleles↗
PubMed2006

Competitive allele-specific short oligonucleotide hybridization (CASSOH) with enzyme-linked immunosorbent assay (ELISA) for the detection of pharmacogenetic single nucleotide polymorphisms (SNPs).

Individualization of drug therapy through genetic testing would maximize the effectiveness of medication and minimize its risks. Recent progress in genetic testing technologies has been remarkable, and they have been applied for the analysis of genetic polymorphisms that regulate drug responses. Clinical application of genetic information to individual health care requires simple and rapid identification of nucleotide changes in clinical settings. We previously reported a novel DNA diagnostic method for detecting single nucleotide polymorphisms (SNPs) using competitive allele-specific short oligonucleotide hybridization (CASSOH) with an immunochromatographic strip. We have developed the method further in order to incorporate an enzyme-linked immunosorbent assay (ELISA) into the final detection step; this enables multiple SNP detection. Special ELISA chips have been fabricated so that disposal of buffer waste is not required and handling procedures are minimized. This method (CASSOH-ELISA) has been successfully applied for the detection of clinically important SNPs in drug metabolism, such as N-acetyltransferase 2, NAT2*6 (590G>A) and NAT*7 (857G>A), and mitochondrial DNA (1555A>G). It would also facilitate point-of-care genetic testing for potentially diverse clinical applications.

Amino-Acid N-Acetyltransferase↗
PubMed2006

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets.

Identification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This "chemical genetics" approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known pharmacological compounds to implicate specific molecular candidates in any chosen biological phenomenon. Taking advantage of a hierarchical structure that can be imposed on drug reagents in a number of fields such as ion transport, neurotransmitter function, metabolism, and cytoskeleton, any assay can be carried out as a binary search algorithm. This inverse drug screen methodology is much more efficient than exhaustive testing of large numbers of drugs, and reveals the identity of a manageable number of specific molecular candidates that can then be validated and targeted using more expensive and specific molecular reagents. Here, we describe the process of this loss-of-function screen and illustrate its use in uncovering novel bioelectrical and serotonergic mechanisms in embryonic patterning. This technique is an inexpensive and rapid complement to existing molecular screening strategies. Moreover, it is applicable to maternal proteins, and model species in which traditional genetic screens are not feasible, significantly extending the opportunities to identify key endogenous players in biological processes.

Biological Assay↗
PubMed1995

An infrared fluorescent dATP for labeling DNA.

Near-infrared fluorescence provides a nonradioactive method of detection with high sensitivity and low background. An infrared fluorophore has been attached covalently to the nucleotide deoxyadenosine triphosphate (dATP) to provide a reagent for enzymatic labeling of various types of DNA molecules and for facilitating their detection with an automated DNA sequencing and analysis system. DNA sequencing reaction products can be labeled internally by performing limited polymerization utilizing infrared-labeled dATP (IR-dATP) as the sole source of adenine deoxynucleotide prior to a dideoxy-specific termination reaction. PCR products can be labeled fluorescently by the addition of limited quantities of IR-dATP to the amplification reaction. This latter strategy has been utilized for detection of short tandem repeat polymorphisms (STRPs) which are useful for gene mapping, genetic diagnostics, forensic analysis, and paternity testing. Restriction fragments can be labeled also by fill-in reactions of appropriate 5' overhangs. Diminutive amounts of such fluorescently labeled DNA molecules can be visualized rapidly and conveniently using infrared detection technology.

Adenosine Triphosphate↗
PubMed1990

A consortium approach to molecular genetic services. Scottish Molecular Genetics Consortium.

The four Scottish university medical genetics centres formed a consortium in 1985 to provide a DNA based service in prenatal diagnosis, carrier detection, and predictive testing for a range of Mendelian disorders. Each centre took sole responsibility for laboratory analyses of an assigned set of disorders, while families continued to be investigated and patients counselled within their own areas. DNA was extracted from relevant tissues in the centre most convenient to the family member and then dispatched to the appropriate laboratory for analysis. Results were interpreted and risks assessed by discussion between laboratory staff and the clinical geneticist in charge of the case. In the first three years of the consortium 92 prenatal diagnoses or exclusion tests were carried out, the majority being for cystic fibrosis (35), Duchenne muscular dystrophy (21), and Huntington's disease (11). Carrier testing was carried out in 271 X linked recessive disorders, the most common indications being Duchenne and Becker muscular dystrophies (198) and haemophilias A and B (48). Predictive testing was attempted in 41 consultants at risk for Huntington's disease, 37 at risk for myotonic dystrophy, and 32 at risk for developing adult polycystic kidney disease. The total of all carrier tests, including those for autosomal recessives, was 543. A consortium or supraregional approach to molecular genetics services has a number of advantages. Constituent laboratories need hold only those probes and enzymes relevant to their assigned disorders and can gain maximum experience with these systems. Scattered families may often be linked into single kinships, thus allowing rapid confirmation of diagnosis when an urgent request is made for a prenatal diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystic Fibrosis↗
PubMed2005

[Hereditary colorectal cancer].

Hereditary syndromes cause approximately 5 to 15% of overall colorectal cancer (CRC) cases. Hereditary CRC is conventionally divided into two major categories: hereditary non-polyposis colorectal cancer (HNPCC) and those related to polyposis syndromes including familial adenomatous polyposis (FAP), Peutz-Jegher syndrome (PJS), and juvenile polyposis (JP). The screening for the cancer and methods of treatment applied to patients with hereditary CRC are quite different from those applied to the general population. The genes responsible for these syndromes has recently identified, as a result, genetic testing has become the most important determining factor in clinical decisions. Germ-line mutation of the APC gene induces FAP, an autosomal dominant disorder, characterized by the development of hundreds to thousands of colonic adenomas. CRC appears in almost all affected individuals by the time they are 50 years of age. An affected individual should undergo colectomy by his/her late teens. Furthermore, according to the findings of genetic testing, at-risk family members also need endoscopic surveillance and surgery. Recently, a mutation on the MYH gene is increasingly being investigated in patients with multiple polyps, and autosomal recessive MYH polyposis is considered to be a new category of polyposis. More common than FAP, HNPCC is caused by germ-line mutations in DNA mismatch repair genes, mainly MLH1 and MSH2. Although there is no polyposis, polyps seem to be more villous and dysplastic and appear to grow rapidly into CRCs. The aggregate lifetime risk of CRC is about 80% for mutation carriers. The risk for other types of cancer, such as endometrial, ovarian, small bowel, and transitional cell cancer, is also increased. The Amsterdam criteria and Bethesda guidelines are the best-known tools for diagnosis and genetic testing, and colectomy followed by endoscopic follow-up is the standard treatment. PJS and JP are reported to be characterized by hamartomatous polyps throughout the GI tract and germ-line mutations in the STK11 gene (PJS) and the DPC4/BMPR1A gene (JP).

Adenomatous Polyposis Coli↗
PubMed2006

Methods and biomarkers for the diagnosis and prognosis of cancer and other diseases: towards personalized medicine.

The rapid development of new diagnostic procedures, the mapping of the human genome, progress in mapping genetic polymorphisms, and recent advances in nucleic acid- and protein chip technologies are driving the development of personalized therapies. This breakthrough in medicine is expected to be achieved largely due to the implementation of "lab-on-the-chip" technology capable of performing hundreds, even thousands of biochemical, cellular and genetic tests on a single sample of blood or other body fluid. Focusing on a few disease-specific examples, this review discusses selected technologies and their combinations likely to be incorporated in the "lab-on-the-chip" and to provide rapid and versatile information about specific diseases entities. Focusing on breast cancer and after an overview of single-nucleotide polymorphism (SNP)-screening methodologies, we discuss the diagnostic and prognostic importance of SNPs. Next, using Duchenne muscular dystrophy (DMD) as an example, we provide a brief overview of powerful and innovative integration of traditional immuno-histochemistry techniques with advanced biophysical methods such as NMR-spectroscopy or Fourier-transformed infrared (FT-IR) spectroscopy. A brief overview of the challenges and opportunities provided by protein and aptamer microarrays follows. We conclude by highlighting novel and promising biochemical markers for the development of personalized treatment of cancer and other diseases: serum cytochrome c, cytokeratin-18 and -19 and their proteolytic fragments for the detection and quantitation of malignant tumor mass, tumor cell turn-over, inflammatory processes during hepatitis and Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis and apoptotic/necrotic cancer cell death.

Animals↗
PubMed1994

Idiopathic pain and depression.

This report summarizes research on the hypothesis that idiopathic chronic pain syndromes and depressive disorders share certain common pathogenetic mechanisms. There is increasing evidence that this may be partly true. Not only do chronic pain syndromes respond to treatment with antidepressants, but there are also striking clinical similarities between these syndromes and depressive syndromes. However, important differences do exist (e.g., the courses of these disorders are usually dissimilar). Family studies show that affective disorders are common in first-degree relatives of patients with idiopathic pain syndromes, but it is impossible to conclude from this that clear-cut genetic factors are of importance. Factors common to both syndromes include common personality traits, shortened rapid eye movements in sleep EEG, hypercortisolaemia and pathological dexamethasone suppression tests, low levels of melatonin in serum and urine and high levels of endorphins and Fraction I in cerebro-spinal fluid. One important common pathogenetic mechanism seems to be disturbances in the serotoninergic system.

Chronic Disease↗
PubMed1983

A new class of rapidly developing mutants in Dictyostelium discoideum: implications for cyclic AMP metabolism and cell differentiation.

Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

Evaluation of two rapid screening assays for the detection of human immunodeficiency virus-1 infection in emergency department patients.

In order to determine the sensitivity and specificity of two rapid human immunodeficiency virus (HIV-1) assays compared with enzyme immunoassay and Western blot and to assess their potential use for routine screening in an emergency department (ED), we analyzed sera from 492 consecutive ED patients using an identity-unlinked design. Sera were analyzed for HIV-1 by standard enzyme-linked immunosorbent assay and Western blot and two rapid assays: the Abbott Testpack HIV-1 (Abbott Labs, Inc, Abbott Park, IL) and the HIV-1 Genie, (Genetic Systems, Seattle, WA). Seroprevalence of HIV-1 among 492 samples was 5.1%. Both rapid assays were easy to perform and required approximately 10 minutes per test. Sensitivity and specificity of both rapid assays were 100% and 99.8%, with positive and negative predictive values of 96.2% and 100%, respectively. It was concluded that both rapid assays showed high concordance with standard enzyme-linked immunosorbent assay and Western blot. Since the ED is often the primary care setting for many patients at risk for HIV-1, the ED may be an optimal site for routine HIV-1 screening. Rapid assay screening may provide the opportunity for timely identification of HIV-1-infected patients, allowing earlier treatment and counseling. However, ethical and practical questions regarding appropriate application of rapid HIV-1 testing in EDs still needs resolution.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

A rabbit model for evaluation of an extracorporeal LDL removal system.

Elevated levels of plasma low density lipoprotein cholesterol (LDL) are associated with an increased incidence of atherosclerosis and its sequelae. A decrease in LDL levels correlates with a reduction in atherosclerosis. Drug therapy and diet have been moderately successful in lowering cholesterol levels, but require significant periods of treatment. Furthermore, a patient with genetically high LDL levels, as in familial hypercholesterolemia, may need a more rapid and reliable method of decreasing serum LDL. The present study was designed to test a device which can rapidly remove LDL from the circulation. The device consists of a filter cartridge filled with semipermeable hollow fibers which have a proprietary acidic polymer (pap) immobilized on the blood/plasma contacting surfaces; this polymer has been reported to selectively bind LDL cholesterol. The device was evaluated in a hypercholesterolemic rabbit model. Reductions in LDL serum concentration, ranging from 1.4-25.3%, occurred in 6/6 studies. All animals survived the procedure. Autopsy results at 10 days showed normal histology. Binding of other proteins (HDL, albumin) were minimal and no hemolysis was observed. Results of this preliminary study demonstrate the feasibility of reducing serum LDL levels with a hollow fiber device.

Animals↗
PubMed2002

Automating data acquisition into ontologies from pharmacogenetics relational data sources using declarative object definitions and XML.

Ontologies are useful for organizing large numbers of concepts having complex relationships, such as the breadth of genetic and clinical knowledge in pharmacogenomics. But because ontologies change and knowledge evolves, it is time consuming to maintain stable mappings to external data sources that are in relational format. We propose a method for interfacing ontology models with data acquisition from external relational data sources. This method uses a declarative interface between the ontology and the data source, and this interface is modeled in the ontology and implemented using XML schema. Data is imported from the relational source into the ontology using XML, and data integrity is checked by validating the XML submission with an XML schema. We have implemented this approach in PharmGKB (http://www.pharmgkb.org/), a pharmacogenetics knowledge base. Our goals were to (1) import genetic sequence data, collected in relational format, into the pharmacogenetics ontology, and (2) automate the process of updating the links between the ontology and data acquisition when the ontology changes. We tested our approach by linking PharmGKB with data acquisition from a relational model of genetic sequence information. The ontology subsequently evolved, and we were able to rapidly update our interface with the external data and continue acquiring the data. Similar approaches may be helpful for integrating other heterogeneous information sources in order make the diversity of pharmacogenetics data amenable to computational analysis.

Databases, Factual↗
PubMed1992

Polymerase chain reaction (PCR) amplification and human leukocyte antigen (HLA)-DQ alpha oligonucleotide typing on biological evidence samples: casework experience.

The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples. The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats. This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated. As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed. The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded. Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed. The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed.

Adolescent↗