Biomedical subjects

Carlos De Brasi

Publications and source records attributed to Carlos De Brasi.

4 recordsLinked to original sources

PubMed2004

Feasibility of a cost-effective approach to evaluate short tandem repeat markers suitable for chimerism follow-up.

BACKGROUND: Precise 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. AIM: To 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. METHOD: Six 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. RESULTS: Four 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 fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH. DISCUSSION: To 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 of Taq polymerase 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. CONCLUSION: The 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.

Bone Marrow Transplantation↗

PubMed2004

A single, multiplex analysis for all relevant activating NRAS gene mutations using heteroduplex generators.

We describe a multiplex polymerase chain reaction (PCR)-based test that detected all relevant NRAS activating mutations using a single PCR followed directly by electrophoresis. The test uses a Universal Heteroduplex Generator (UHG) to detect exon-2 (codon 61) NRAS mutations in multiplex with an UHG for exon-1 (codons 12 and 13). The method differentiated all 19 relevant mutations in these exons and showed a mutation independent sensitivity of approximately 6%. The sensitive, specific detection of all NRAS activating mutations using this single rapid test represents a minimum workload and could be applied readily for large-scale screening and for routine analysis.

Base Sequence↗

PubMed2003

Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology.

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.

Codon↗

PubMedDate not supplied

Incidence of BCL-2 gene rearrangements in Argentinean non-Hodgkin lymphoma patients: increased frequency of breakpoints outside of MBR and MCR.

The t(14;18)(q32;q21) is the most frequent cytogenetic abnormality observed in follicular lymphoma (FL), but also occurs in diffuse large B-cell lymphoma (DLBCL). We have evaluated the frequency of this translocation in 106 Argentinean non-Hodgkin lymphoma (NHL) patients: 83 with diagnosis of FL and 23 with DLBCL. Nested (N) and long-distance PCR (LD-PCR) approaches were used. By N-PCR, a total of 42 (51%) FL cases showed BCL-2 rearrangement: 28 (34%) for major breakpoint region (MBR) and 14 (17%) for minor cluster region (mcr). By LD-PCR, additional 23 (28%) new positive cases were found: 10 (12%) for MBR and 13 (16%) for mcr. These data make a total of 65 (78%) positive cases for BCL-2 gene rearrangements. In DLBCL cases, N-PCR detected two (9%) cases with the MBR breakpoint and one (4%) with mcr. Seven (30%) new positive cases by LD-PCR were found: four (17%) for MBR and three (13%) for mcr, showing a total of 10 (43%) positive cases. Thus, both FL and DLBCL had high frequencies of breakpoints located between MBR and mcr clusters. Our N-PCR results in FL (51%; 95% CI, 40-62%) showed statistical differences with respect to the pooled data from USA (P < 0.0001) and overlapped with the frequencies from Asia and Europe. In DLBCL, no significant differences with respect to the literature were found. This data support the idea that FL may be a heterogeneous malignancy with distinct molecular pathogenesis and suggest that the geographic differences may be related with the distribution of breakpoints that are widely spread throughout the sequence stretch between MBR and mcr.

Adult↗