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Results for “genetic test utilization”

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PubMed1995

An improved Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation.

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7.9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10(5) cfu micrograms-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus, it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.

DNA, Bacterial↗

PubMed1980

Identification and mapping of a second proline permease Salmonella typhimurium.

In this paper we demonstrate the existence of a second proline permease, gene proP, in Salmonella typhimurium. Uptake assays demonstrate that this second proline permease has 5 to 10% the uptake rate of the putP permease, the cell's major proline permease, when assayed at 20 microM proline. Genetic mapping by Hfr and P22-mediated genetic crosses placed the second proline permease gene at 92 min on the S. typhimurium genetic map, near the genes for melibiose utilization. F'-mediated complementation tests indicated that Escherichia coli also has the proP gene.

Chromosome Mapping↗

PubMed1982

Genes for l-sorbose utilization in Escherichia coli.

Amongst forty wild strains of Escherichia coli, nine used L-sorbose as a source of carbon and energy and two mutated to use it. Laboratory strains K12, B and C were L-sorbose-negative. Genes for L-sorbose utilization (sor+) were transferred to K12 from six wild strains; genes conferring the mutable phenotype were also transferred. All were cotransducible with metA at 90 min on the linkage map. The most probable gene order was met ace sor pgi mal. Complementation tests identified two genes for L-sorbose utilization. Genetical evidence showed that the catabolite repressor protein of K12 exerted positive control over sor+ genes introduced into K12. The genes for phosphofructokinase (pfkA), the phosphocarrier protein (ptsH) and phosphotransferase enzyme I (ptsI) were required for utilization of L-sorbose. The frequency of transduction of sor+ was low when selection was made for sor+, because L-sorbose partially inhibited the growth of both L-sorbose-negative strains and K12 (sor+) strains. Uridine, thymidine and sorbitol each annulled the inhibition of growth and increased the frequency of transduction of sor+.

Escherichia coli↗

PubMed2003

Conversion technology and its role in genetic testing of inherited diseases.

Identification of germline mutations in a sensitive and specific manner presents a continuing challenge. A major contributing factor to this is that humans are diploid and therefore mutations in one allele are often masked by the normal sequence present on the other copy of the chromosome. Mutation analysis on haploid templates (one copy of a chromosome), rather than on diploid templates (both copies of a chromosome), overcomes this problem and obscured mutations are unmasked. Conversion technology converts a sample from diploid to haploid state by isolating individual alleles in somatic cell hybrids. From each sample, a series of stable hybrids is generated that contains the human chromosome complement in the haploid state. This article describes conversion technology and its applications. The utility of this technique in increasing the sensitivity of genetic testing has been demonstrated for the predisposition to hereditary nonpolyposis colorectal cancer and it is proposed that a similar approach may be applicable to many different diseases.

Animals↗

PubMed2005

Single nucleotide polymorphisms in clinical genetic testing: the characterization of the clinical significance of genetic variants and their application in clinical research for BRCA1.

Clinical genetic testing is increasingly employed in the medical management of cancer patients. These tests support a variety of clinical decisions by providing results that indicate risk for future disease, confirmation of diagnoses, and more recently, therapeutic selection and prognosis. Most genetic variation detected during clinical testing involves single nucleotide polymorphisms (SNPs). Continued advances in the technologies of genetic analyses make these tests increasingly sensitive, cost-effective and timely, which contribute to their increased utilization. Conversely, it has proven difficult to characterize the clinical significance of genetic variants that do not obviously truncate the open reading frames of genes. These genetic variants of uncertain clinical significance diminish the value of genetic test results. This article highlights a variety of approaches that have emerged from research in diverse disciplines to solve the problem, including the application of information about common SNPs in multiple methods to better characterize clinically uncertain variants. Hereditary breast/ovarian cancer, and in particular BRCA1, provides a framework for this discussion. BRCA1 is particularly interesting in this respect since clinical genetic testing by direct DNA sequencing for over 50,000 patients in North America has revealed approximately 1500 genetic variants to date. This large data set combined with the clinical significance of BRCA1 have resulted in research groups selecting BRCA1 as a preferred gene to evaluate novel methods in this field. Finally, the lessons learned through work with BRCA1 are highly applicable to many other genes associated with cancer risk.

Breast Neoplasms↗

PubMed2005

Genomic medicine and thrombotic risk: who, when, how and why?

Major advances in Human Genome research could significantly change the clinical medical practice, providing new possibilities for both diagnosing and treating common pathologies. Many genetic tests are now commercially available for predicting future risk of common disorders. However, genetic testing has potential benefits but also limitations for the patients, and it should not be used to 'screen' the general population. Diagnostic assays for a predisposition of both venous and arterial thrombosis are among the most requested genetic tests in molecular diagnostics laboratories. However, there is considerable uncertainty as to how this information should be utilized in patient management. Both the medical community and the patients need to obtain accurate information concerning the appropriate use of genetic testing. The purpose of this article is to discuss the usefulness and the practical applications of thrombotic genetic testing in order to define which patients should be tested for both venous and arterial thrombotic risk as well as to have an acceptable cost/benefit ratio and to prevent patients' anxiety.

Ethics, Medical↗

PubMed2005

How can the evaluation of genetic tests be enhanced? Lessons learned from the ACCE framework and evaluating genetic tests in the United Kingdom.

Advances in genetic technology are increasing the availability of genetic tests, not only for rare single gene disorders, but also for common diseases such as breast and colo-rectal cancer. Before there can be widespread uptake of these tests, they must be evaluated to confirm the benefits of their use. But how should genetic tests be evaluated, given the speed at which new tests are emerging? One highly influential approach is the analytic validity, clinical validity, clinical utility and ethical, legal and social issues (ACCE) framework, which has provided a benchmark for the evaluation of genetic tests. The approach has been adopted and adapted by the United Kingdom Genetic Testing Network, with the help of the Public Health Genetics Unit in Cambridge, to evaluate new genetic tests for use in the National Health Service. We discuss a number of conceptual, methodological, and practical issues concerning the evaluation of genetic tests, based on lessons learned from applying the ACCE framework and from the UK experience, and make a number of recommendations to further strengthen the evaluation of genetic tests.

Genetic Carrier Screening↗

PubMed2003

Exploratory subsetting of autism families based on savant skills improves evidence of genetic linkage to 15q11-q13.

OBJECTIVE: Autism displays a remarkably high heritability but a complex genetic etiology. One approach to identifying susceptibility loci under these conditions is to define more homogeneous subsets of families on the basis of genetically relevant phenotypic or biological characteristics that vary from case to case. METHOD: The authors performed a principal components analysis, using items from the Autism Diagnostic Interview, which resulted in six clusters of variables, five of which showed significant sib-sib correlation. The utility of these phenotypic subsets was tested in an exploratory genetic analysis of the autism candidate region on chromosome 15q11-q13. RESULTS: When the Collaborative Linkage Study of Autism sample was divided, on the basis of mean proband score for the "savant skills" cluster, the heterogeneity logarithm of the odds under a recessive model at D15S511, within the GABRB3 gene, increased from 0.6 to 2.6 in the subset of families in which probands had greater savant skills. CONCLUSIONS: These data are consistent with the genetic contribution of a 15q locus to autism susceptibility in a subset of affected individuals exhibiting savant skills. Similar types of skills have been noted in individuals with Prader-Willi syndrome, which results from deletions of this chromosomal region.

Autistic Disorder↗

PubMed1998

A functional myo-inositol catabolism pathway is essential for rhizopine utilization by Sinorhizobium meliloti.

Rhizopine (L-3-O-methyl-scyllo-inosamine) is a symbiosis-specific compound found in alfalfa nodules induced by specific Sinorhizobium meliloti strains. It has been postulated that rhizobial strains able to synthesize and catabolize rhizopine gain a competitive advantage in the rhizosphere. The pathway of rhizopine degradation is analysed here. Since rhizopine is an inositol derivative, it was tested whether inositol catabolism is involved in rhizopine utilization. A genetic locus required for the catabolism of inositol as sole carbon source was cloned from S. meliloti. This locus was delimited by transposon Tn5 mutagenesis and its DNA sequence was determined. Based on DNA similarity studies and enzyme assays, this genetic region was shown to encode an S. meliloti myo-inositol dehydrogenase. Strains that harboured a mutation in the myo-inositol dehydrogenase gene (idhA) did not display myo-inositol dehydrogenase activity, were unable to utilize myo-inositol as sole carbon/energy source, and were unable to catabolize rhizopine. Thus, myo-inositol dehydrogenase activity is essential for rhizopine utilization in S. meliloti.

Amino Acid Sequence↗

PubMed2004

Odour identification as a marker for postoperative cognitive dysfunction: a pilot study.

The onset of postoperative cognitive dysfunction is delayed in some elderly surgical patients and the condition may persist. Increasing age is a risk factor for both postoperative cognitive dysfunction and dementia. Major surgery may unmask an underlying dementia; we report findings of a pilot study designed to inform this hypothesis. Impaired odour identification ability is a characteristic of the preclinical phase of some neuro-degenerative diseases. This pilot study was designed to establish whether odour identification deficit is a marker for postoperative cognitive dysfunction, particularly in elderly patients genetically predisposed to develop late-onset Alzheimer's disease. We found no association between odour identification ability and postoperative cognitive dysfunction in 53 patients aged 62-86, undergoing major non-cardiac surgery under general anaesthesia. Our pilot study showed that a high number of patients genetically predisposed to develop Alzheimer's disease is required to test the utility of odour identification as a marker for postoperative cognitive dysfunction.

Aged↗

PubMed2001

Practice parameter: diagnosis of dementia (an evidence-based review). Report of the Quality Standards Subcommittee of the American Academy of Neurology.

OBJECTIVE: To update the 1994 practice parameter for the diagnosis of dementia in the elderly. BACKGROUND: The AAN previously published a practice parameter on dementia in 1994. New research and clinical developments warrant an update of some aspects of diagnosis. METHODS: Studies published in English from 1985 through 1999 were identified that addressed four questions: 1) Are the current criteria for the diagnosis of dementia reliable? 2) Are the current diagnostic criteria able to establish a diagnosis for the prevalent dementias in the elderly? 3) Do laboratory tests improve the accuracy of the clinical diagnosis of dementing illness? 4) What comorbidities should be evaluated in elderly patients undergoing an initial assessment for dementia? RECOMMENDATIONS: Based on evidence in the literature, the following recommendations are made. 1) The DSM-III-R definition of dementia is reliable and should be used (Guideline). 2) The National Institute of Neurologic, Communicative Disorders and Stroke--AD and Related Disorders Association (NINCDS-ADRDA) or the Diagnostic and Statistical Manual, 3rd edition, revised (DSM-IIIR) diagnostic criteria for AD and clinical criteria for Creutzfeldt--Jakob disease (CJD) have sufficient reliability and validity and should be used (Guideline). Diagnostic criteria for vascular dementia, dementia with Lewy bodies, and frontotemporal dementia may be of use in clinical practice (Option) but have imperfect reliability and validity. 3) Structural neuroimaging with either a noncontrast CT or MR scan in the initial evaluation of patients with dementia is appropriate. Because of insufficient data on validity, no other imaging procedure is recommended (Guideline). There are currently no genetic markers recommended for routine diagnostic purposes (Guideline). The CSF 14-3-3 protein is useful for confirming or rejecting the diagnosis of CJD (Guideline). 4) Screening for depression, B(12) deficiency, and hypothyroidism should be performed (Guideline). Screening for syphilis in patients with dementia is not justified unless clinical suspicion for neurosyphilis is present (Guideline). CONCLUSIONS: Diagnostic criteria for dementia have improved since the 1994 practice parameter. Further research is needed to improve clinical definitions of dementia and its subtypes, as well as to determine the utility of various instruments of neuroimaging, biomarkers, and genetic testing in increasing diagnostic accuracy.

Dementia↗

PubMed2002

Evaluation of factors affecting individual assignment precision using microsatellite data from horse breeds and simulated breed crosses.

Assignment tests have been utilized to investigate population classification, measure genetic diversity and to solve forensic questions. Using microsatellite data from 26 loci genotyped in eight horse breeds we examined how population differentiation, number of scored loci, number of scored animals per breed and loci variability affected individual assignment precision applying log likelihood methods. We found that both genetic differentiation and number of scored loci were highly important for recognizing the breed of origin. When comparing two and two breeds, a proportion of 95% of the most differentiated breeds (0.200 < or = FST < or = 0.259) could be identified scoring only three loci, while the corresponding number was six for the least differentiated breeds (0.080 < or = FST < or = 0.139). An identical proportion of simulated breed crosses, differentiated from their parental breeds by FST estimates in the range 0.050-0.069, was identified when scoring 12 loci. This level of source identification was not obtained for the less differentiated breed crosses. The current data further suggested that population sample size and locus variability were not critical for the assignment precision as long as moderately large sample sizes (> or = 20 animals per population) and fairly variable loci were used.

Animals↗

PubMed2003

Linkage and association studies of QTL for nuclear families by mixed models.

The transmission disequilibrium test (TDT) has been utilized to test the linkage and association between a genetic trait locus and a marker. Spielman et al. (1993) introduced TDT to test linkage between a qualitative trait and a marker in the presence of association. In the presence of linkage, TDT can be applied to test for association for fine mapping (Martin et al., 1997; Spielman and Ewens, 1996). In recent years, extensive research has been carried out on the TDT between a quantitative trait and a marker locus (Allison, 1997; Fan et al., 2002; George et al., 1999; Rabinowitz, 1997; Xiong et al., 1998; Zhu and Elston, 2000, 2001). The original TDT for both qualitative and quantitative traits requires unrelated offspring of heterozygous parents for analysis, and much research has been carried out to extend it to fit for different settings. For nuclear families with multiple offspring, one approach is to treat each child independently for analysis. Obviously, this may not be a valid method since offspring of one family are related to each other. Another approach is to select one offspring randomly from each family for analysis. However, with this method much information may be lost. Martin et al. (1997, 2000) constructed useful statistical tests to analyse the data for qualitative traits. In this paper, we propose to use mixed models to analyse sample data of nuclear families with multiple offspring for quantitative traits according to the models in Amos (1994). The method uses data of all offspring by taking into account their trait mean and variance-covariance structures, which contain all the effects of major gene locus, polygenic loci and environment. A test statistic based on mixed models is shown to be more powerful than the test statistic proposed by George et al. (1999) under moderate disequilibrium for nuclear families. Moreover, it has higher power than the TDT statistic which is constructed by randomly choosing a single offspring from each nuclear family.

Asthma↗

PubMed1997

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive "staircase"), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.

Actins↗

PubMed2001

Cardiac performance in inbred rat genetic models of low and high running capacity.

1. Previous work demonstrating that DA inbred rats are superior to COP inbred rats in aerobic treadmill running capacity has indicated their utility as genetic models to explore this trait. We tested the general hypothesis that intermediate phenotypes of cardiac function and calcium metabolism are responsible for the difference in capacity between these strains. 2. Logical cardiac trait differences were estimated at a tissue (isolated papillary muscle), cellular (isolated left ventricular cells), and biochemical level of organization. 3. DA hearts were found to give significantly higher values than COP hearts for: (1) maximal developed tension (38.3 % greater), and rates of tension change in contraction (61 %) or relaxation (59 %) of isolated papillary muscle, (2) fractional shortening (50 %), amplitude of the Ca(2+) transient (78.6 %), and caffeine-induced release of Ca(2+) from the sarcoplasmic reticulum (SR; 260 %) in isolated ventricular myocytes, and (3) Na(+),K(+)-ATPase activity of isolated myocytes (17.3 %). 4. Our results suggest that these trait differences may prove useful for further studies into the genes responsible for natural variations in both ventricular function and aerobic endurance capacity. Understanding the genetic basis of aerobic capacity will help define the continuum between health and disease.

Animals↗

PubMedDate not supplied

Telomerase as a target for cancer immunotherapy.

Telomerase is the ribonucleoprotein that enables cancer and stem cells to maintain their telomeres, resulting in unlimited proliferative potential. The catalytic component of telomerase in humans, hTERT, is upregulated in nearly 90% of all cancers, making it the most widely expressed marker of malignancy. With the exception of germ cells and stem cells, hTERT is undetectable in somatic human tissues. Together, these properties make telomerase a leading candidate for cancer therapy. Various therapies have been tested in tissue culture and in mouse models utilizing genetic, pharmacological, and immunological approaches. The purpose of this review is to critically examine the role of hTERT in cancer immunotherapy by providing a comparison of the current experiments and a proposal for an innovative method utilizing DNA vaccination.

CD4-Positive T-Lymphocytes↗

PubMed1999

Molecular genetic basis of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma is a complex genetic disease. As might be expected for a malignancy that is rather homogeneous in clinical presentation and behavior, a distinct subset of genes are found to be genetically inactivated in a majority of the tumors. A yet larger subset of genes experiences genetic inactivation at much lower frequencies. The latter subset could solely reflect a somewhat trivial genetic heterogeneity of the tumor, but more likely will represent the initial insights into pathways whose more widespread importance will be shown in future work. Familial pancreatic cancer susceptibility underlies a significant fraction of the overall incidence. Genetic testing is feasible for many of the causative genes, although the clinical utility remains unsettled. The precursor lesion for pancreatic cancer shares some of the genetic lesions of the more advanced invasive stage, and follows a stepwise progression model both histologically and genetically. Genes Chromosomes Cancer 26:1-12, 1999.

Adenocarcinoma↗

PubMedDate not supplied

Providing information at the point of care: educational diagnostic reports from a genetic testing service provider.

Strategies to facilitate the provisioning of genetic health-care services by primary care physicians will improve access to these services for the average patient while making the most efficient use of limited human and financial resources within the health-care system. Genetic laboratories have become a major source of information and consultation for clinicians ordering genetic tests. This article describes the development and evaluation of a program to provide physician-directed information from a molecular testing facility to assist physicians in selecting and counseling patients and interpreting genetic test results. Semi-structured telephone interviews were used to gather physicians' opinions about the utility of the program and the way in which the information was used in their practice. Forty-three percent of those interviewed were unfamiliar with some of the information provided, with test methodology and sensitivity/specificity most often identified as novel information. Fifty-two percent of pediatric specialists were unfamiliar with some aspect of the information Sheet, despite being the highest consumers of testing services in this sample. Pediatricians and pediatric specialists also rated the initiative highest in terms of its usefulness in their practice, followed by genetic specialists. Overall, physicians confirmed the utility of the program as an educational tool for themselves, and for other non-patient educational activities in which they are involved.

Attitude of Health Personnel↗