摘要:

The pace of development in novel technologies that promise improvements in the early diagnosis of disease is truly impressive. One such technology at the forefront of this revolution is mass spectrometry. New capabilities in mass spectrometry have provided the means for the development of proteomics, and the race is on to find innovative ways to apply this powerful technology to solving the problems faced in clinical medicine. One area that has garnered much attention over the past few years is the use of mass spectral patterns for cancer diagnostics.The use of these so-called ‘proteomic patterns’ for disease diagnosis relies fundamentally on the pattern of signals observed within a mass spectrum rather than the more conventional identification and quantitation of a biomarker such as in the case of cancer antigen-125- or prostate-specific antigen. The inherent throughput of proteomic pattern technology enables the analysis of hundreds of clinical samples per day. Currently, there are two primary means by which proteomic patterns can be acquired, surface-enhanced laser desorption/ionization (SELDI) and an electrospray ionization (ESI) method that has been popularized under the name, OvaCheck™. In this review, an historical perspective on the development of proteomic patterns for the diagnosis of early-stage cancers is described. In addition, a critical assessment of the overall technology is presented with an emphasis on the steps required to enable proteomic pattern analysis to become a viable clinical tool for diagnosing early-stage cancers.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundPrecise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients.AimTo set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT.MethodSix dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients.ResultsFour markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67–0.88) under our experimental system. A sensitivity of 0.8–1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescencein situhybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH.DiscussionTo our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage ofTaqpolymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient.ConclusionThe data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundIn the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identifyMYCNgene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma.AimThe aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma,MYCNamplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer.MethodsDNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region andMYCNgene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control andArabidopsis thalianaDNA was spotted to control unspecific hybridization. Fluorescencein situhybridization analysis was also performed to validate results from the microarray CGH.ResultsBothMYCNamplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detectMYCNamplification.DiscussionOur results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundThree mutations (R702W, G908R, and 1007fs) within theCARD15gene have been identified as independent risk factors for the development of Crohn’s disease (CD). Virtually all studies investigating the occurrence of these mutations in patients with CD have used separate PCR-based methods to screen patient DNA, here we describe a novel multiplex amplification refractory mutation system (ARMS) assay that allows the simultaneous detection of R702W, G908R, and 1007fs, and a fourthCARD15variant, P268S, at a fraction of the cost of the pre-existing genotyping assays.MethodsAllele-specific primer sets were designed for eachCARD15variant, optimized separately for annealing temperature and MgCl2and then multiplexed. The mutant- and wild-type-specific primers were split across two tubes so that each multiplex reaction was internally controlled for amplification failure. An additional primer pair specific to β2-microglobulin was included as an independent control for DNA quality. The specificity of each primer set was tested using positive controls that had been validated by sequencing, and the robustness of the final ARMS assay was assessed by genotyping 111 Caucasian patients with inflammatory bowel disease (IBD).ResultsThe specificity of each primer set was confirmed using a sequence validated positive control for each of the fourCARD15variants. Of the 111 DNA samples screened with our ARMS assay, a clearCARD15genotype was obtained for 109 patients.Discussion and conclusionsGiven the potential predictive value of R702W, G980R, and 1007fs, a robust genotyping method for these variants would be of considerable value both in diagnostic and research settings. Our ARMS assay only takes 3–4 hours to perform once DNA has been extracted and requires only 1U ofTaqDNA polymerase, making it a rapid, reliable, and cost-effective alternative to currentCARD15genotyping methods.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundDifferences in gene expression are frequently encountered in malignant tissues, and have been intensively studied as they can reflect different experimental or clinical conditions. Quantification of the often subtle changes in messenger RNA content is performed through comparison with the expression of endogenous controls. The appropriate choice of these endogenous controls (e.g. housekeeping genes) is critical for meaningful quantitative RNA analysis. The most important characteristics of housekeeping genes are that they are present in all cells and that their expression levels remain relatively constant in different experimental conditions. However, no single housekeeping gene always manifests stable expression levels under all experimental conditions. Therefore, it is necessary to characterize the suitability of various housekeeping genes to serve as internal RNA controls under particular experimental conditions where transcription effects are being tested.AimIt was the aim of this study to determine the validity of a number of housekeeping genes for their use as internal standards in cancer research.MethodsThe expression of the housekeeping genes porphobilinogen deaminase (PBGD) and mitochondrial ATP synthase 6 (mATPsy6), were compared with the expression of the more commonly used glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We examined a number of cell lines and tumor versus matched normal tissue samples using real-time quantitative (RTq)-PCR.ResultsOur findings suggest that in cell lines, all three of the studied housekeeping genes can be used as an internal control. When comparing tumor tissue samples with matched normal tissue samples, we validated mitochondrial ATPsy6 (mATPsy6) as the best choice for a housekeeping gene.ConclusionSince gene expression studies are becoming increasingly important in the clinical environment, especially in cancer diagnosis and treatment, the use of an reliable housekeeping gene in these studies to normalize gene expression is essential. We conclude that a bad choice of housekeeping gene may lead to errors when interpreting experiments involving quantitation of gene expression. Our study demonstrated the usefulness of mATPsy6 as an endogenous control when comparing tumor tissue samples with normal tissue samples.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundApproximately one-third of new cases of Duchenne muscular dystrophy (DMD) can be attributed to sporadically arising new mutations, however in the majority of cases the DMD mutation has been inherited from the mother. These female carriers can have either a constitutive or mosaic mutation.AimThe aim of this study was to determine the segregation of the at-risk haplotype and to find a deletion in the dystrophin gene of patients.MethodWe analyzed individuals from two families with a history of DMD in order to predict the carrier status ofrelatedfemales. In one of these cases the mother had two affected sons, while in the other one son and two grandchildren were affected; therefore we predict that the mother would be an obligatory carrier.ResultsHaplotype analysis of theDMDloci revealed that in the two families both the healthy and affected brothers had inherited the same X maternal chromosome. However, the affected brother carried a deletion, which was absent in the unaffected sibling.ConclusionThese findings suggested that the mothers in the two families were germline mosaics for theDMDgene. The results of this study demonstrate the usefulness of the methodology that combine the haplotype analysis with the identification of the mutation in order to detect hidden germline mosaicisms and, thus, improve genetic counseling.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

摘要:

BackgroundAs genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5–9 have been developed as reference materials for the detection ofTP53mutations.AimThe goal of this study was to validate the reference materials in providing quality assurance for the detection ofTP53mutations in clinical specimens.MethodsWe studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5–9 for mutation detection.ResultsOf the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned.ConclusionsMutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST)TP53reference panel inTP53mutation detection in clinical tissue specimens.

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS

ISSN:1084-8592    

出版商:ADIS    

年代:2004

数据来源: ADIS