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PubMed2004

Development and integration of molecular genetic tests into clinical practice: the US experience.

The issues that arise in the development of genetic tests for prediction and diagnosis are described in the context of the authors' experience as laboratory directors in the USA. The goal is to identify gaps and weaknesses in the test validation process and to define the pivotal issues. Variables that influence a laboratory director's decision to develop a particular molecular genetic assay, including motivation, economics, intellectual property and the regulatory environment, are described. Issues of clinical and analytic validation are discussed, providing examples of tests with both good (cystic fibrosis carrier screening) and poor (apolipoprotein E genotyping for Alzheimer's disease) clinical utility. The decision-making process that occurs during the considered transition of a research-based molecular genetic assay into routine use in the clinical laboratory is summarized. Different factors will be weighted differently depending on the nature of the disease being tested, the complexity of its gene and mutations, the available technical platforms, potential regulatory and intellectual property restrictions, and whether the proposed test is to be offered by an academic or a commercial laboratory.

Alzheimer Disease↗
PubMed2006

Application of molecular diagnostic methods to penaeid shrimp diseases: advances of the past 10 years for control of viral diseases in farmed shrimp.

The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.

Animal Diseases↗
PubMed2005

Effects of Bcl-2 levels on Fas signaling-induced caspase-3 activation: molecular genetic tests of computational model predictions.

Fas-induced apoptosis is a critical process for normal immune system development and function. Although many molecular components in the Fas signaling pathway have been identified, a systematic understanding of how they work together to determine network dynamics and apoptosis itself has remained elusive. To address this, we generated a computational model for interpreting and predicting effects of pathway component properties. The model integrates current information concerning the signaling network downstream of Fas activation, through both type I and type II pathways, until activation of caspase-3. Unknown parameter values in the model were estimated using experimental data obtained from human Jurkat T cells. To elucidate critical signaling network properties, we examined the effects of altering the level of Bcl-2 on the kinetics of caspase-3 activation, using both overexpression and knockdown in the model and experimentally. Overexpression was used to distinguish among alternative hypotheses for inhibitory binding interactions of Bcl-2 with various components in the mitochondrial pathway. In comparing model simulations with experimental results, we find the best agreement when Bcl-2 blocks the release of cytochrome c by binding to both Bax and truncated Bid instead of Bax, truncated Bid, or Bid alone. Moreover, although Bcl-2 overexpression strongly reduces caspase-3 activation, Bcl-2 knockdown has a negligible effect, demonstrating a general model finding that varying the expression levels of signal molecules frequently has asymmetric effects on the outcome. Finally, we demonstrate that the relative dominance of type I vs type II pathways can be switched by varying particular signaling component levels without changing network structure.

Apoptosis↗
PubMed2001

[The present status and future prospect of the molecular diagnostic tests].

Assays for DNA or RNA sequences to diagnose infectious, neoplastic and genetic diseases have been widely used through recent progress in the molecular biology and biotechnology, and are now essential in care of patients under the advanced medicine through earlier and more accurate diagnosis. Automated systems have been developed for amplification and detection of nucleic acid sequence for infectious agents, using various nucleic acid amplification technology such as PCR. A fully automated PCR system and automated extraction of specific sequence for infectious agents such as hepatitis C virus RNA has been developed. These automated systems have provided improvement of not only assay efficiency but also quality control of the tests and have contributed to the standardization of them. Importance of development of systems for quality assessment and laboratory accreditation has been emphasized, particularly in those that still have been performed with manual methods. Based on the information on the genome sequence as the outcome of the human genome project, functions of genes and proteins have been studied by post-genomics such as expression profiling using DNA microarray, proteomics, single nucleotide polymorphisms analysis, coupled with bioinformatics. Along with advances in pharmacogenomics, these studies have raised the prospect of the development of tests for individualized medicine based on genetic information such as those predicting individual susceptibility to diseases for prevention and responsiveness to drugs for choice of treatment. For practice of such medicine, each genetic information and tests for it must be carefully evaluated and determined whether it is appropriate for cost-effective medicine through contributions to efficient process of decision-makings on patient care for prevention or avoidance of diseases and thus to cost savings.

Cytogenetic Analysis↗
PubMed2004

The -omics era and its impact.

OBJECTIVE: To review the advances in clinically useful molecular biologic techniques and to identify their applications, as presented at the 12th Annual William Beaumont Hospital DNA Symposium. DATA SOURCES: The 7 manuscripts submitted were reviewed and their major findings were compared with literature on the same or related topics. STUDY SELECTION: Manuscripts address the use of molecular techniques in the detection of severe acute respiratory syndrome (SARS) and bacterial ribosome mutations, which may lead to ribosome-targeted drug resistance; pharmacogenomics as a clinical laboratory service and example of warfarin dosing using CYP2C9 mutation analysis; definition of the potential of cytosine arabinoside incorporation into DNA to disrupt transcription using an in vitro model of oligonucleotides; use of laser capture microdissection to isolate solid tumor cells free of nontumor cells; and molecular methods used to classify lymphomas. DATA SYNTHESIS: Two current issues related to the use of molecular tests in the clinical laboratories are (1) decentralization of molecular-based testing to a variety of nonmolecular laboratories and (2) need for wider acceptance of molecular-based testing through its incorporation in clinical practice guidelines. Molecular methods have had a major impact on infectious disease through the rapid identification of new infectious agents, SARS, and the characterization of drug resistance. Pharmacogenomics identifies the genetic basis for heritable and interindividual variation in response to drugs. The incorporation of the nucleoside analog, cytosine arabinoside, into DNA leads to local perturbation of DNA structure and reduces the ability of transcription factors to bind to their specific DNA binding elements as measured by electrophoretic mobility shift assays. Laser capture microdissection of tumor cells can provide an adequate number of cells for whole genome amplification. Gene expression microassay profiles of various lymphomas have modified classification systems and predict prognosis and response to therapy. CONCLUSIONS: The current -omics era will continue to emphasize the use of microarrays and database software for genomic, transcriptomic, and proteomic screening to search for a useful clinical assay. The number of molecular pathologic techniques will expand as additional disease-associated mutations are defined.

Animals↗
PubMed2003

Hepatitis C virus RNA tests: performance attributes and their impact on clinical utility.

The diagnostic market is driven by the burden of disease in the population and the ease or difficulty of disease diagnosis. The efficacy of available therapeutics determines the need for monitoring. Hepatitis C virus currently affects approximately 3% of the world's population, although the overall response rate to the best available therapies is 56%. Research regarding hepatitis C virus remains elusive due to lack of an efficient cell culture system. Diagnosis and monitoring of active hepatitis C virus infection therefore rely on sophisticated molecular tests. This review will focus on current molecular tests for hepatitis C virus RNA and the performance attributes that these tests require for accurate diagnosis and monitoring of infection.

Hepacivirus↗
PubMed1998

[Presumptive tests and molecular hybridization for the identification of untypable strains of Streptococcus pneumoniae].

Five different methods for identification of pneumococci (optochine susceptibility, bile solubility, Slidex Pneumo-kit, Phadebact Pneumococcus test, AccuProbe DNA test) were evaluated with a total of 280 Streptococcus pneumoniae non typable strains. 189 strains were identified as pneumococci according to the AccuProbe test results. Among these, 180 strains (95.2%) were optochine sensitive (d > or = 12 mm). Bile solubility was seen in 125 (66.1%) of the pneumococci. Immunological identifications were respectively positive for 67 and 56 among 140 strains. By comparison with the DNA/RNA reassociation method, the poor sensitivities and specificities of the presumptive identification tests are actually demonstrated for pneumococcal non typable strains. Thus, the AccuProbe DNA test is seen as the only adequate method for identification of such strains.

Bacterial Typing Techniques↗
PubMed1999

Molecular diagnostic testing in Charcot-Marie-Tooth disease and related disorders. Approaches and results.

The inherited neuropathies of the peripheral nervous system are clinically and genetically a heterogeneous group of disorders. Molecular genetic studies have made major breakthroughs in unraveling the underlying gene defects, and DNA diagnosis can now be offered to a large number of families with distinct forms of hereditary peripheral neuropathies. With the currently available technology, however, molecular genetic diagnosis still remains a labor-intensive and costly procedure. We have developed an algorithm for mutation screening based on clinical phenotype, electrophysiological findings, and the relative frequencies of mutations in the distinct peripheral myelin genes.

Charcot-Marie-Tooth Disease↗
PubMed2003

Ab initio quality one-electron properties of large molecules: development and testing of molecular tailoring approach.

The development of a linear-scaling method, viz. "molecular tailoring approach" with an emphasis on accurate computation of one-electron properties of large molecules is reported. This method is based on fragmenting the reference macromolecule into a number of small, overlapping molecules of similar size. The density matrix (DM) of the parent molecule is synthesized from the individual fragment DMs, computed separately at the Hartree-Fock (HF) level, and is used for property evaluation. In effect, this method reduces the O(N(3)) scaling order within HF theory to an n.O(N'(3)) one, where n is the number of fragments and N', the average number of basis functions in the fragment molecules. An algorithm and a program in FORTRAN 90 have been developed for an automated fragmentation of large molecular systems. One-electron properties such as the molecular electrostatic potential, molecular electron density along with their topography, as well as the dipole moment are computed using this approach for medium and large test chemical systems of varying nature (tocopherol, a model polypeptide and a silicious zeolite). The results are compared qualitatively and quantitatively with the corresponding actual ones for some cases. This method is also extended to obtain MP2 level DMs and electronic properties of large systems and found to be equally successful.

Algorithms↗
PubMed2006

Molecular genetic testing for familial hypercholesterolemia in the Netherlands: a stepwise screening strategy enhances the mutation detection rate.

Familial hypercholesterolemia (FH) has been identified as a major risk factor for coronary vascular disease and is associated with mutations in the low-density liporotein receptor (LDLR) and apolipoprotein B (APOB) gene. The molecular basis of FH in the Dutch population is well understood. Approximately 160 different LDLR and APOB gene defects have been identified with a panel of 9 LDLR gene and 1 APOB gene frequently occurring mutations accounting for approximately 30% of all clinically diagnosed FH cases. As molecular diagnosis of FH is becoming increasingly widely applied, a variety of mutation detection rates is reported, ranging from as low as 30% and up to 80%. This variability appears to depend on the clinical criteria applied to identify patients with FH and on the strategies and methodologies used for mutation screening. In this study we describe the application of a stepwise screening approach, combining different methodologies, to detect mutations of the LDLR gene and APOB gene in 1465 patients with FH. A mutation was found in approximately 44% of the patients, which demonstrates that this is an effective strategy for the molecular diagnosis of FH.

Adolescent↗
PubMed2000

Detection of bladder cancer by semi-automated microsatellite analysis of urine sediment.

Patients with transitional-cell carcinoma (TCC) require careful follow-up due to the high risk of recurrence. Cystoscopy and biopsy are reliable but invasive, while urine cytology is plagued by low sensitivity. It has recently been shown that allelic abnormalities detected by microsatellite analysis of DNA extracted from urine can be used to diagnose TCC with high reliability. As this analysis by classic techniques is unfeasible in a clinical setting, we performed a pilot study to determine the possibility of applying quick DNA extraction methods with laser detection and computer-based analysis of 15 fluorescently labeled PCR amplified microsatellites to detect molecular anomalies in urine sediment in 25 TCC follow-up patients. Of the eighteen cases with recurrent TCC, 14 (78%) were positive by the molecular test whereas only eight (44%) were detected by cytology. Of the seven patients with negative cystoscopy, one resulted positive by the molecular test and had recurrent TCC six-months later. Thus, this microsatellite analysis correctly predicted the clinical diagnosis in 84% (21/25) of cases, compared to 60% by cytology. The application of these semi-automated procedures allows the analysis of 18 samples with 15 markers in one day, encouraging a more expedient introduction into routine clinical use.

Aged↗
PubMed2002

Sib-parentage testing using molecular markers when parents are unknown.

The formulae for computing the so-called Sib Index using codominant alleles for (1) full-sib and (2) half-sib parentage are given. Hypothesis testing is based on the distribution of conditional likelihood ratio or Bayes' factor. Thresholds for rejecting the null hypothesis and P-values were obtained in function of the number of alleles and their frequency distributions. Simulations showed that a relatively low number of marker systems (e.g. 20) are enough to accept the hypothesis of sib parentage with a reasonable power for usual significance levels, but that a higher number would be necessary if full-sib against half-sib parentage is the contrast to be carried out. The effect of sampling variation on the allele frequencies on power calculations is also analysed.

Alleles↗
PubMed2004

Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia.

Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease, is associated with an unstable expansion of a GAA trinucleotide repeat in the first intron of the frataxin gene on chromosome 9q13. Unequivocal molecular characterization of the FRDA triplet expansion requires the use of different PCR protocols to amplify normal and mutated alleles combined with Southern blotting analysis to accurately size the expansion. Nevertheless, expansion detection by PCR may be somewhat problematic in heterozygous individuals. The purpose of this study was to evaluate triplet repeat primed PCR (TP PCR) as a screening method for FRDA diagnosis in the diagnostic laboratory. Fifty-four cases referred either to confirm the diagnosis of FRDA or to detect carrier status were re-evaluated by the TP PCR method. The TP PCR assay correctly identified the FRDA status in all 54 individuals tested including homozygous expansions (9 individuals), heterozygous expansions (20 individuals), and non-carriers (25 individuals). Results showed 100% concordance with those obtained by Southern blot analysis. TP PCR allowed us to identify the expanded alleles or to demonstrate their absence in DNA samples where conventional PCR procedures failed to give a reliable result. TP PCR represents an additional valuable tool for mutation detection in FRDA patients and carriers, but also can be used as screening test in a diagnostic laboratory.

Alleles↗
PubMed2001

Genetic counseling and risk communication services of newborn screening programs.

OBJECTIVES: Newborn screening test results labeled "positive" can have uncertain implications for parents, especially when false-positive results occur or when heterozygous infants are detected using molecular tests for sickle cell hemoglobinopathy or cystic fibrosis. This study surveyed communication services across state newborn screening programs. METHODS: We surveyed newborn screening programs to identify current communication practices and the methods used for quality assessment. Two successive survey instruments with fixed-answer and free-answer questions were distributed to screening program follow-up coordinators or similar designated officials associated with 52 states and territories. RESULTS: Replies from 46 respondents (89% response rate) revealed that regional newborn screening programs vary widely in their approaches to counseling. Of the 46 respondents, 35 (76%) answered that they "routinely" provide counseling services to families of affected infants. Depending on the disease, an average of approximately one-half that number provide counseling after false-positive results or for heterozygous infants. Most respondents advocate nondirective counseling more than direct advice. Most programs reported that counseling was usually done by subspecialist physicians or specially trained nurses and counselors. Respondents reported a perception that the "quality" of counseling by these professionals is better than counseling by primary care physicians. Few programs reported systems for assessing quality assurance of counseling. CONCLUSIONS: Newborn screening programs in the United States vary widely with regard to counseling practices, and no best practices are currently evident. Few programs provide counseling quality assurance. Further study and advocacy is needed to optimize communication services, preferably before implementation of molecular tests arising as a result of the Human Genome Project.

Communication↗