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PubMed1998

Escherichia coli molecular phylogeny using the incongruence length difference test.

Molecular phylogeny of the species Escherichia coli using the E. coli reference (ECOR) collection strains has been hampered by (1) the absence of rooting in the commonly used phenogram obtained from multilocus enzyme electrophoresis (MLEE) data and (2) the existence of recombination events between strains that scramble phylogenetic trees reconstructed from the nucleotide sequences of genes. We attempted to determine the phylogeny for E. coli based on the ECOR strain data by extracting from GenBank the nucleotide sequences of 11 chromosomal structural and 2 plasmid genes for which the Salmonella enterica homologous gene sequences were available. For each of the 13 DNA data sets studied, incongruence with a nonnucleotide whole-genome data set including MLEE, random amplified polymorphic DNA, and rrn restriction fragment length polymorphism data was measured using the incongruence length difference (ILD) test of Farris et al. As previously reported, the incongruence observed between the gnd and plasmid gene data and the whole-genome data was multiple, indicating numerous horizontal transfer and/or recombination events. In five cases, the incongruence detected by the ILD test was punctual, and the donor group was identified. Congruence was not rejected for the remaining data sets. The strains responsible for incongruences with the whole-genome data set were removed, leading to a "prior-agreement" approach, i.e., the determination of a phylogeny for E. coli based on several genes, excluding (1) the genes with multiple incongruences with the whole genome data, (2) the strains responsible for punctual incongruences, and (3) the genes incongruent with each other. The obtained phylogeny shows that the most basal group of E. coli strains is the B2 group rather than the A group, as generally thought. The D group then emerges as the sister group of the rest. Finally, the A and B1 groups are sister groups. Interestingly, the most primitive taxon within E. coli in terms of branching pattern, i.e., the B2 group, includes highly virulent extraintestinal strains with derived characters (extraintestinal virulence determinants) occurring on its own branch.

Enzymes↗

PubMed1994

Issues in molecular genetic testing of individuals with suspected early-onset familial Alzheimer's disease.

The identification of mutations in the amyloid precursor protein (APP) gene associated with the presence of early-onset familial Alzheimer disease (AD) raises the possibility of their practical clinical application, at least in some circumstances, in the diagnostic assessment for AD. As a stimulus for discussion, a hypothetical, illustrative case vignette is presented. A 48-year-old man, concerned about recent memory loss and with a family history of early-onset AD, requested testing for the APP717 Val-->Ile mutation, previously identified in his relatives affected with AD. Whether the testing should be undertaken is considered in the context of the current interpretation of potential test results as well as the competency of the individual who requested the test to provide informed consent. Informed consent includes an understanding of the foreseeable risks and benefits associated with disclosure of test results. Although molecular genetic testing in particular individuals, such as the man described herein, could be appropriate, it should not be interpreted to apply in general at this stage to individuals suspected of having AD. In view of a number of caveats, including the genetic heterogeneity of AD, which significantly limits the sensitivity and specificity of the currently available genetic tests, further research and discussion is strongly recommended before widespread introduction of molecular genetic testing for individuals with suspected AD.

Alzheimer Disease↗

PubMed2005

Molecular diagnostic testing for inherited thrombophilia using Invader.

Physicians in the United States and Europe began testing patients who had idiopathic thrombotic events for inherited risk factors in 1990s. The College of American Pathologists (CAP) offered proficiency testing for molecular genetic screening for thrombophilia in 1997. Today, a hypercoagulable workup including screening for inherited thrombophilia defects is becoming part of the standard of care in many parts of the world (1). Who, what, and when to test continue to be controversial and challenging questions (2); however, laboratories developing new or improved mutation detection methodologies have used the most commonly screened inherited thrombophilia polymorphism, factor V Leiden (R506Q), for many years (3). In the most recent CAP survey (MGL-A 2003), the most commonly employed method used in one-third of all participating clinical laboratories testing for factor V Leiden and prothrombin G20210A was the non-PCR-based method called Invader, developed by Third Wave Technologies. The remainder of the clinical laboratories reported testing for these variants using PCR-restriction fragment length polymorphism (RFLP), allele-specific PCR, and allele-specific hybridization. Emerging mutation detection technologies include DNA resequencing approaches such as pyrosequencing (4) fluorescence polarization detection (5), genotyping on microelectronic DNA chips like Nanogen's nanochip (6), and oligonucleotide hybridization with photocrosslinking (7). Invader technology is currently a medium-throughput, 96-well plate format assay that is sufficient for most hospital clinical laboratories. Although this assay format is not currently performed in a microarray format, Invader is amenable to performance on a solid support; specifically, the reaction can be performed on the surface of microspheres and the resulting fluorescence measured using flow cytometry (8). This chapter presents the method as well as some suggestions for utilizing the Invader system for mutation/polymorphism screening in general and for thrombophilia testing in particular.

Factor V↗

PubMed1996

Pitfalls in the interpretation of molecular diagnostic tests.

Application of the tools of molecular genetics has vastly increased the power of genetic diagnosis, but also raises significant challenges in interpretation. Molecular tests can be used for diagnosis of an affected individual or determination of carrier status. Either direct mutation analysis or linkage analysis is used in various disorders. Major pitfalls in interpretation of direct tests include mistaking polymorphisms for mutations, inaccurate prognostication based on identification of a mutation, and misinterpretation of the implications of not finding a mutation. Interpretation of linkage tests must take account of the accuracy of proband diagnosis, interpretation of family relationships, and genetic recombination. Cognizance of these issues is vital to avoidance of clinical errors and must be incorporated into genetic counseling.

Diagnostic Tests, Routine↗

PubMed2001

Effect of mutagen-induced cell lethality on the dose response of germline mutations.

Molecular tests for mutations require a sample of tissue from which DNA is extracted, to determine the presence or absence of one or more mutations per sample. To ensure mutation fixation each sample must consist of an equal number of cells that have had one or more DNA replications. In an in vivo test, surviving stem cells compensate to give the same number of cells per sample, leaving as the only evidence for stem cell lethality the increase in mutants of clonal origin because the mutant clone developed from a population of fewer stem cells. A problem is that an increase in mutagen dose increases stem cell death, resulting in a decreased number of surviving target cells, thus giving a downward bias of samples with one or more mutations per sample. To compare in vivo tests with molecular tests we will use as a model system the sex-linked recessive lethal (SLRL) test for germ cell mutations in Drosophila melanogaster. Spermatogonia cells in male larvae were exposed to ENU and mutations detected in sperm cells from adults. The same SLRL data were analyzed by two methods: (1) The conventional analysis of SLRL data, in which each mutation of a cluster of mutations of common origin was counted. (2) An analysis was used to simulate a sample for molecular analysis by determining mutations per male with an equal size sample of progeny per male. With this second analysis a correction factor is required based on the change in cluster size of mutants of common origin.

Animals↗

PubMed2002

[Recent advances in molecular diagnostic tests].

Recent advances in molecular biotechnologies have facilitated laboratory use of molecular diagnostic tests. Automated systems for amplification and detection, and lately for extraction, of nucleic acid sequences have been developed. The automated systems have allowed improvement of not only assay efficiency but also quality control of the tests. The information on the genome sequence from the human genome project has been studied to elucidate functions of genes and proteins and the clinical significance of nucleic acid sequences with post-genomics such as expression profiling using DNA microarray, proteomics, and single nucleotide polymorphism analysis, in conjunction with bioinformatics. Now the challenge is the development and application of these new technologies as clinical diagnostic tools. Design of a diagnostic array or panel must be developed with defined sequences based on interpretation of a huge quantity of experimental data to meet clinical demands. There is a need for development of generally available instruments that are inexpensive, practical and more importantly reproducible and reliable.

Computational Biology↗

PubMed2000

Molecular Papanicolaou tests in the twenty-first century: molecular analyses with fluid-based Papanicolaou technology.

OBJECTIVE: This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system. STUDY DESIGN: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase-polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides. RESULTS: Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen. CONCLUSION: It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test.

Carcinoembryonic Antigen↗

PubMed2001

[Molecular diagnostic tests in hematologic diseases].

Molecular diagnostic tests are widely performed in managing hematologic malignancies such as leukemia and lymphoma. In this article, we review the present application and problems of the tests. Karyotyping is performed at diagnosis of all kinds of hematologic malignancies. This method needs dividing cells as samples and skilled experts. Fluorescence in situ hybridization(FISH) analysis using cells in interphase is performed, for example, to monitor the effect of interferon on chronic myelogenous leukemia patients. The weak point of this method is that approximately 2% of false-positive cells are inevitable. Southern blot method is used for clonal analysis in some disease, for example, adult T-cell leukemia/lymphoma. Polymerase chain reaction(PCR) method using genomic DNA is performed for limited types of diseases such as lymphoma with bcl-2/IgH fusion gene. Reverse transcription(RT)-PCR method can detect fusion gene transcripts with high sensitivity. This method is useful for detecting minimal residual diseases after chemotherapy or bone marrow transplantation. To perform quantitative analysis, real-time PCR or competitive PCR must be done. In the near future, new technology such as gene expression profiling analysis using DNA microarrays or spectral karyotyping(SKY) method will be used in clinical practice.

Blotting, Southern↗

PubMed2002

ACOG technology assessment. Genetics and molecular diagnostic testing. Number 1, July 2002. American College of Obstetrics and Gynecology.

Human genetics and molecular testing is playing an increasingly important role in obstetric and gynecologic practice. It is essential that obstetricians and gynecologists are aware of the advances in our understanding of genetic disease and the fundamental principles of molecular testing and genetic screening as genetics is integrated into routine medical practice. In the future, elucidation of the genetic basis for reproductive disorders, common diseases, and cancer with improved high throughput technology for genetic testing will expand testing opportunities and influence treatment options and prevention strategies.

Female↗

PubMed2007

Adaptation of the ultrasensitive HIV-1 p24 antigen assay to dried blood spot testing.

Implementation of molecular tests for the assessment of pediatric HIV-1 infection in resource-limited countries is difficult because of technical complexity and costs. Alternatives like the ultrasensitive HIV-1 p24 antigen enzyme-linked immunosorbent assay have therefore been proposed. We have now adapted this test to dried blood spot (DBS) plasma p24 antigen (p24). High background activity was recognized as originating from endogenous peroxidase and eliminated by H2O2 quenching. The assay was evaluated with 72 pediatric specimens from Tanzania and with 210 pediatric or adult specimens from Switzerland. A real-time polymerase chain reaction assay for DBS DNA and/or plasma RNA identified HIV-1 infection in 38 Tanzanian children. HIV-1 subtypes included 18 C, 9 A1, 8 D, 1 AC, 1 J-like, and 1 unidentified. The detection rates for the different assays were as follows: DBS-p24, 32 (84%) of 38 samples; DBS DNA, 30 (79%) of 38 samples; plasma-p24, 23 (85%) of 27 samples; and plasma RNA, 30 (100%) of 30 samples. False-negative DBS-p24 was associated with subtype D (P < 0.01). DBS-p24 detection for non-D subtypes was 93% (95% confidence interval: 81% to 99%), and for subtype C, it was 94% (95% confidence interval: 76% to 99%). Specificity among 193 HIV-negative DBS samples was 100%. Correlation of DBS-p24 and plasma-p24 concentrations was excellent (R = 0.83, P < 0.0001). DBS-p24 is thus a promising alternative to molecular tests for HIV-1 in subtype C regions. It should now be evaluated in large studies of children for accurate assessment of diagnostic sensitivity.

Adolescent↗

PubMed2003

[Genetic tests for molecular markers of cancer].

Based on recent advancements in biotechnology and molecular oncology, genetic tests for molecular markers have been widely used for diagnosis and therapeutic monitoring in patients with hematological malignancies. However, incorporation of such tests has been limited in the care of patients with non-hematopoietic malignancies. The reasons for this include difficulty in sampling of the tumor tissue, and a lack of evidence for cost-effectiveness in the test utilization. A practical approach is the use of bodily fluids, such as blood, sputum, urine and feces, as specimens for early detection and monitoring of tumor-derived nucleic acid sequences. The prospect has been raised for development of tests for individualized medicine based on genetic information such as prediction of responsiveness to chemotherapeutic agents for selection of treatment, which will be promoted by gene expression profiling using DNA microarray.

Biomarkers, Tumor↗

PubMed1999

Quality assurance in molecular genetic testing laboratories.

CONTEXT: Specific regulation of laboratories performing molecular genetic tests may be needed to ensure standards and quality assurance (QA) and safeguard patient rights to informed consent and confidentiality. However, comprehensive analysis of current practices of such laboratories, important for assessing the need for regulation and its impact on access to testing, has not been conducted. OBJECTIVE: To collect and analyze data regarding availability of clinical molecular genetic testing, including personnel standards and laboratory practices. DESIGN: A mail survey in June 1997 of molecular genetic testing laboratory directors and assignment of a QA score based on responses to genetic testing process items. SETTING: Hospital-based, independent, and research-based molecular genetic testing laboratories in the United States. PARTICIPANTS: Directors of molecular genetic testing laboratories (n = 245; response rate, 74.9%). MAIN OUTCOME MEASURE: Laboratory process QA score, using the American College of Medical Genetics Laboratory Practice Committee standards. RESULTS: The 245 responding laboratories reported availability of testing for 94 disorders. Personnel qualifications varied, although all directors had doctoral degrees. The mean QAscore was 90% (range, 44%-100%) with 36 laboratories (15%) scoring lower than 70%. Higher scores were associated with test menu size of more than 4 tests (P = .01), performance of more than 30 analyses annually (P = .01), director having a PhD vs MD degree (P = .002), director board certification (P = .03), independent (P <.001) and hospital (P = .01) laboratories vs research laboratory, participation in proficiency testing (P<.001), and Clinical Laboratory Improvement Amendment certification (P = .006). Seventy percent of laboratories provided access to genetic counseling, 69% had a confidentiality policy, and 45% required informed consent prior to testing. CONCLUSION: The finding that a number of laboratories had QA scores that may reflect suboptimal laboratory practices suggests that both personnel qualification and laboratory practice standards are most in need of improvement to ensure quality in clinical molecular genetic testing laboratories.

Certification↗

PubMed1999

The impact of molecular diagnostic tests on patient outcomes.

In order for the benefits of molecular pathology to outweigh its inherent costs, the procedures must be integrated into the assessment of the total disease management to realize the true impact of their financial and clinical outcomes. Major financial and patient outcome benefits are achievable from molecular testing by the ability to reduce the use of less-sensitive and less-specific tests and cause a decrease the use of unnecessary diagnostic procedures and ineffective therapies. In this review, the financial and patient care determinants of outcomes for molecular-based testing of disease predisposition, screening, early detection, and directed therapy are presented.

Genetic Techniques↗

PubMed2004

Molecular genetic testing for inherited disorders.

This review focuses on molecular genetic testing for heritable disorders. Molecular genetic testing has generated tremendous interest because of the scientific and social hype surrounding the Human Genome Project, the accelerating rate of gene discovery, the quick transition of scientific discoveries into clinically applicable genetic tests and the unique nonmedical applications of genetic testing. All of these developments have been superimposed upon increasing individual autonomy in medical care and the pervasive information-seeking behavior of the Information Age.

DNA Mutational Analysis↗

PubMed1997

Molecular genetic testing for adult-onset disorders: the evolving laboratory, physician, patient interface.

Molecular genetics is providing the power to predict and diagnose a variety of diseases. This is an evolving field, with new information being gained at a very rapid rate. Ultimately the molecular tools should be available to prevent and/or cure a number of diseases with genetic etiology. The molecular genetics laboratory is playing a pivotal role in effecting the utilization of this technology. However, molecular genetic testing is in many ways unlike other clinical laboratory testing. The process of molecular genetic testing and counseling is complex. The laboratory offering molecular genetic testing must be able to assess pedigrees, decide the proper test to perform and provide interpretation of the tests results obtained. Although standards governing the use of high-complexity genetic tests are evolving it is clear that the laboratory offering these tests will have to have on staff personnel trained specifically in molecular genetics. The challenge will be to stay abreast of evolving technology as well as standards and recommendations governing the use of these tests. We will review the process of molecular genetic testing and the issues that must be considered by laboratories who offer these tests.

Accreditation↗

PubMed2003

Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.

Positive control materials for clinical molecular genetic testing applications are currently in critically short supply or non-existent for many genetically based diseases of public health importance. Here we demonstrate that anonymous, residual, clinical blood samples are potential sources of viable lymphocytes for establishing Epstein-Barr virus (EBV)-transformed blood lymphocyte cell lines. We attempted to transform 34 residual blood samples, and analyzed transformation success with respect to sample age, anticoagulant, storage temperature, volume, hemolysis, and patient age and sex. In univariate analysis, sample age was significantly associated with transformation success (P = 0.002). The success rate was 67% (6 of 9) for samples 1 to 7 days old, 38% (3 of 8) for samples 8 to 14 days old and 0% for samples 15 to 21 (0 of 11) days old. When we controlled for sample age in multivariate logistic regression, anticoagulant and storage temperature approached significance (P = 0.070 and 0.087, respectively; samples in acid citrate dextrose (ACD) and refrigerated samples were more likely to transform). Based on these findings, we suggest that samples collected in either ACD or ethylene diamine tetraacetic acid, and up to 14 days old (refrigerated) or 7 days old (stored ambient), are reasonable candidates for EBV transformation. The transformation rate for samples that met these criteria was 63% (10 of 16). Implementation of this process could help alleviate the shortage of positive control materials for clinical molecular genetic testing.

Adult↗

PubMed2005

Technical standards and guidelines: molecular genetic testing for ultra-rare disorders.

These standards and guidelines are designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical molecular geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the laboratory record the rationale for any significant deviation from these standards and guidelines.

Genetic Testing↗

PubMed1999

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

BACKGROUND AND PURPOSE: X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. METHODS: The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." RESULTS: The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. CONCLUSIONS: Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage.

Adult↗