ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundAmong the well characterized X-linked conditions causing mental retardation, mutations in the methyl-CpG-binding protein 2 gene (MECP2) in Xq28 have been found in up to 85% of patients with Rett syndrome, a neurologic disorder which, in addition to other symptoms, severely affects higher cognitive functions in females. Mutations in theMECP2gene are involved in a broad spectrum of phenotypes from classical Rett syndrome to mild intellectual difficulties in females and neonatal encephalopathy in males. Recently, mutations in theMECP2gene were reported in males with non-specific mental retardation suggesting that defects inMECP2could be responsible for up to 2% of X-linked mental retardation.MethodsWe screened by denaturing high-pressure liquid chromatography the entire coding region and flanking intronic sequences of theMECP2gene in a cohort of 354 mentally retarded males found negative for an expansion across the FRAXA CGG repeat and in a family in which a boy and his sister were mentally retarded.ResultsWe identified mainly silent polymorphisms within theMECP2gene, together with four sequence alterations of unknown significance, i.e. three missense mutations (T197M, T228S, and P376S) and one substitution at position –19 in intron 3 (378-19delT). Further familial investigations allowed us to ruled out a pathogenic effect for the intronic variant, the T228S and the P376S missense mutations.ConclusionsThese results confirm thatMECP2mutations in males are far more rare than initially thought and call for a careful evaluation of the pathogenicity of theMECP2missense mutations identified in mentally retarded males before genetic counseling is proposed to the relatives.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundReal-time PCR is a useful method for detecting and quantifying bacterial DNA in clinical samples. DNA extraction is a crucial step when performing quantitative PCR.MethodsWe compared three methods, QIAamp®1DNA Mini kit, MagNAPure™ LC DNA Isolation Kit II together with PickPen™ magnetic particle transfer device, and KingFisher®genomic DNA purification Kit with KingFisher®mL instrument, for purification ofStreptococcus pneumoniaeDNA from 50 nasopharyngeal swab samples, collected into skim milk-tryptone-glucose-glycerin medium. Pneumococcal DNA was detected and quantified by real-time PCR and results were compared to culture findings.ResultsThe 22 (44%) pneumococcal culture-positive specimens were all positive by PCR regardless of DNA extraction method used, except that one KingFisher-extracted sample was positive only when repeatedly tested. Additionally, 71%, 57%, and 82% of the culture-negative samples were positive by real-time PCR when DNA was extracted by QIAamp, MagNAPure-PickPen, and KingFisher methods, respectively. The number of genome equivalents detected by real-time PCR varied, but was mainly low in culture-negative samples. The sensitivities of culture and real-time PCR were hence compared by analyzing different dilutions of a pneumococcal suspension. Real-time PCR detected significantly higher numbers of genome equivalents than the numbers of bacteria detected by culture.ConclusionsThe results indicate that the DNA extraction method used for quantitative PCR should be evaluated and that real-time PCR is more sensitive than bacterial culture for detecting pneumococcus in nasopharyngeal swab samples.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundInterferon-β (IFNβ) has proven to be an important advance in the therapy of multiple sclerosis (MS), but optimal markers for bioactivity have not been identified. To accurately measure bioactivity in MS patients treated with IFNβ, we developed and tested a real-time reverse transcriptase (RT)-PCR assay for gene expression ofMxA, an IFNβ-induced gene in the peripheral blood of patients treated with IFNβ.MethodsWe compared IFNβ-treated patients with MS to controls in expression ofMxArelative to the house-keeping gene,GAPDH. 2′-5′oligoadenylate synthetase (OAS) gene expression was also tested by real-time RT-PCR on RNA from the same patient specimens. Anti-IFNβ antibody was measured by ELISA and a cytopathic effect assay.ResultsSeven of 54 patients were found to have complete loss of bioactivity.MxAexpression correlated well withOASexpression. All patients with lost bioactivity had high levels of binding antibodies or neutralizing antibodies.ConclusionsThis is the first demonstration that a real-time RT-PCR assay can be used to monitor therapy with interferons. These data identifyMxAmRNA as an excellent biomarker for INFβ action on the IFN receptor, and clarify the relationship between anti-IFNβ antibodies and bioactivity in patients with MS treated with IFNβ.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundIt is widely known that the efficiency of fluorescencein situhybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of anALKFISH probe, during the validation of its diagnostic utility.MethodsWe applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals.ResultsWe found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times.ConclusionsThe difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

AimThe Factor V Leiden mutation (G1691A) is a clinically important polymorphism that results in an increased risk of thrombosis. The goal of this study was to compare a temperature gradient capillary electrophoresis (TGCE) platform for the detection of Factor V gene mutations to a conventional restriction fragment length polymorphism (RFLP) assay.MethodsThree hundred and four samples were analyzed by both TGCE and a common clinicalMnlI/RFLP assay. Concordance of results between the two assays was observed for 302/304 (99.3%) of the samples.ResultsAll of the Leiden mutants (23/23, 100%) were identified by TGCE. Of the two discrepant results, one was caused by low peak heights in the TGCE output data and was easily rectified by the addition of a minimum peak height threshold. The second discrepancy resulted from the presence of a G→A transition 95bp downstream of the Leiden mutation site. This polymorphism represents a previously unreported alteration of the Factor V gene.ConclusionsThe TGCE assay is less labor-intensive and has a higher throughput capacity than theMnlI/RFLP assay. TGCE is a less specific assay than theMnlI/RFLP assay that allows for the detection of novel polymorphisms, but also creates the need for all positive TGCE results to be confirmed by an alternate method such as sequencing. Our results demonstrate that TGCE is a highly sensitive method for mutation detection and has utility for mutation discovery analysis.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

BackgroundDuring the course of multiple sclerosis (MS) intrathecal oligoclonal IgGs are present in the cerebrospinal fluid (CSF). The intracellular human pathogenChlamydia pneumoniaemay play a role either as a causative pathogenetic agent in the disease, orC. pneumoniae-infected MS patients could be immunologically less able to clear the agent from the central nervous system (CNS).MethodsCSF samples were studied in 100 individuals – 70 MS patients and 30 age-matched controls with other neurological diseases. CSF was taken by lumbal puncture; cell cultures were performed by the cell vial technique, followed by a 4-day incubation at 37°C. A nested PCR was performed.ResultsC. pneumoniaewas detectable in the CSF of only 2.9% of the MS patients and none of control patients (with no significant difference between the MS patients and controls). IgG antibodies were positive in only 1.43% of the MS patients and 3.33% of the controls. IgA antibodies were positive in 6.66% of the control patients and none of the patients were positive for IgM antibodies. There was no statistically significant difference between the two groups of patients with respect to the three antibody classes.ConclusionsThe results confirm the high leave of controversy surrounding a possible link betweenC. pneumoniaeand MS, and the matter requires further thorough investigation.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

IntroductionMutations at the codon 6 of theβ-globingene (hemoglobin [Hb] S and HbC) can be routinely identified by various methods and prenatal diagnosis has been available to affected families for several years. However, the presence of maternal cells in fetal samples constitutes a serious potential source of prenatal misdiagnosis and most methods currently used to detect maternal contamination are based on the analysis of highly polymorphic loci. In addition, these methods are labor intensive and time consuming and risk carry-over contamination.MethodWe describe here a one-step method for mutation detection that uses fluorescent hybridization probes with melting curve analysis for both simultaneously prenatal diagnosis of sickle cell disease and potential maternal contamination.ResultsRetrospective and prospective prenatal diagnosis studies (conducted in 20 and 50 cases, respectively), using both the regular procedure and real-time PCR assay show perfect concordant results. We show in addition, that as little as 5% maternal contamination can be detected and that genotype determinations are unambiguous.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

In the US, one in every eight women will develop breast cancer in her lifetime. Despite the advances made in treating breast cancer, the causal mechanisms underlying this disease have yet to be fully elucidated; 85% of breast cancer cases occur sporadically without any known genetic mutation.Too little is known about the pathogenesis of breast cancer for primary prevention to be feasible in the near- to mid-term. Secondary prevention through screening offers an alternative that has been widely adopted. For decades, breast self-examination has been touted as a technique for the early identification of breast cancer. However, it has been recently suggested that this technique is a waste of time and resources for both doctors and patients.Mammography finds breast cancer earlier than breast self-examination, and will reduce the risk of death from breast cancer by approximately 30% in women over 50 years old. Mammography is limited in that cancer, like breast tissue, appears white on the x-ray; therefore lesions may be difficult to detect in women with very dense breasts, and a tumor may not cast a significant shadow until it is quite large. Some cancers are so aggressive that they can spread quickly, before routine screening can detect them. Despite these limitations, mammography is still viewed as the best tool currently available for screening and early diagnosis.Improved methods to detect and diagnose breast cancer early, when it is most curable, are required if a significant impact on morbidity and mortality from breast cancer is to be made. Various new and innovative technologies are being investigated for improving the early detection and diagnosis of breast cancer. About 85% of breast cancers begin in the milk ductal system of the breast. As cancer develops in the breast, abnormalities occur, including atypical hyperplasia, ductal carcinomain situ, and invasive breast carcinoma. Thus, the early screening of ductal cells can provide a parallel benefit to the ‘Pap’ smear, which is used virtually universally to identify the abnormal cells that can lead to cervical cancer. Two technologies to monitor for atypical ductal epithelial cells are Cytyc Corporation’s FirstCyte™ Ductal Lavage system and Nastech Pharmaceutical Company’s Mammary Aspiration Cytology Test.Matritech, Inc. is searching for biomarkers linked to breast cancer. Researchers at Matritech have detected the presence of nuclear matrix protein (NMP) in the blood of women at the early stage of breast cancer, which is absent in the blood of healthy women, as well as those with fibroadenoma, a benign breast disease. NMP66 has been selected as a marker for further development and clinical trials of a test for use in the detection and monitoring of women with, or at risk for, breast cancer have been initiated.Technologies developed by the US Department of Defense are under investigation as breast cancer screening. Advanced Image Enhancement, Inc. has licensed naval sonar technology for digital image enhancement of mammograms. New thermography applications are also being investigated in two separate projects sponsored by the US Department of Defense using military thermal surveillance tools adapted for cancer detection. Both are enhancements of older thermal imaging technology based on the principle that heat equates to unwanted activity, in the case of breast cancer, abnormal cell proliferation.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS

摘要:

Molecular diagnostics is a rapidly evolving area, both in the identification of new molecular markers of disease and treatment response, and in the development of technologies to apply them in the clinic. To help keep you up to date with the latest advances, this section of the journal brings you abstracts selected from recently published issues of Adis journals, covering innovative technologies and clinical applications of molecular markers for the diagnosis and targeted treatment of human disease.

ISSN:1084-8592    

出版商:ADIS    

年代:2003

数据来源: ADIS