Browsing by Author "Rivera Fuentes, Juan Carlos"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Identificación del gen CEBPα en pacientes con leucemia mieloide aguda y su potencial uso clínico.
    (Universidad de Concepción, 2023) Rivera Fuentes, Juan Carlos; Aguayo Tapia, Claudio Rodrigo; Chandía, Mauricio
    Acute myeloblastic leukemia (AML) arises from the accumulation of mutations in hematopoietic stem cells, resulting in a disruption of cell differentiation, apoptosis, and increased proliferation. One of the notable molecular alterations involves point mutations in the CEBPα gene, which have been associated with a favorable prognosis in terms of overall survival and event-free survival. Patients with CEBPα mutations tend to exhibit better treatment responses compared to those without these mutations. Although the clinical guide for leukemic patients issued by the Ministry of Health acknowledges the molecular study of the CEBPα gene in adults, it is currently unavailable for this age group as well as pediatric patients in any public clinical laboratory specializing in molecular biology. This limitation primarily stems from the absence of standardized polymerase chain reaction (PCR) techniques for amplifying the CEBPα gene. The gene's coding region presents a high guanine (G) and cytosine (C) base content (> 65%) and includes a trinucleotide repeat sequence, posing challenges in the amplification process. Successful amplification of the CEBPα gene necessitates a comparison and selection of DNA extraction techniques and the standardization of PCR conditions based on the gene's characteristics. Developing a comprehensive protocol encompassing amplification and sequencing will enable the characterization of the CEBPα gene in AML patients. An observational cross-sectional cohort study was conducted during the first semester of 2021. A non-probability convenience sampling method was employed, and an initial sample of three AML-free volunteers was included. Subsequently, DNA extraction was performed on peripheral blood and bone marrow samples from ten AML patients. The extraction techniques were evaluated based on DNA concentration, purity, cost, and time. PCR was then conducted to amplify the CEBPα gene in both healthy individuals and AML patients, using both published and newly designed primers. The amplification protocol underwent evaluation by adjusting annealing temperatures and incorporating adjuvants such as DMSO at varying concentrations. Agarose gel electrophoresis was utilized to assess the integrity and amplified DNA. Finally, the amplified regions were sequenced using the Sanger method, establishing a comprehensive protocol for studying the CEBPα gene. Among the evaluated extraction techniques, the salt extraction method employed on concentrated leukocyte samples demonstrated the most optimal performance. Significant statistical differences (p<0.005) in DNA concentration were observed compared to the column technique. PCR standardization successfully accomplished the amplification of the complete coding region of the CEBPα gene using a single set of primers, as well as fragment amplification using two sets of primers. The optimal annealing temperatures for all primer pairs were determined to be 62 and 64 °C, and the use of 3% and 8% DMSO facilitated the generation of single bands in agarose gel electrophoresis. The sequencing method employed was the Sanger method, which yielded optimally sequenced products for the amino terminal region, enabling the elaboration of a consensus sequence. Standardizing the DNA extraction, PCR, and sequencing techniques for studying the CEBPα gene will provide valuable diagnostic information for AML patients, equipping clinicians with improved tools to make informed decisions regarding appropriate therapy in this context.
Síguenos en...