RESUMO
The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.
Assuntos
Processamento Alternativo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , Translocação Genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Biologia Computacional/métodos , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Éxons , Meia-Vida , Humanos , Íntrons , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Transdução de Sinais , Processos EstocásticosRESUMO
The RUNX1-RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1-RUNX1T1 fusion gene.
Assuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Translocação Genética , Transformação Celular Neoplásica/genética , Mapeamento Cromossômico , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1 , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
BACKGROUND: Currently, it is widely accepted that one of the crucial players in adult leukemic transformation is the RUNX1 gene. However, there is little data available regarding whether mutations in this gene also contribute to pediatric leukemia, especially in childhood myeloid malignancies. Therefore we made a decision to screen patients with pediatric myeloid neoplasias for the presence of RUNX1 mutations in their samples. PROCEDURES: Patients (n = 238) with diagnoses of de novo acute myeloid leukemia (AML) (n = 198), de novo myelodisplastic syndrome (MDS) (n = 16), therapy-related AML (n = 9), juvenile myelomonocytic leukemia (JMML) (n = 15) were included in this study. All patients were Belarusians between the ages of 0 and 18 years. RESULTS: The frequency of RUNX1 point mutations in the total group of patients with de novo AML was 3% and de novo MDS was 15%. Cooperation of point mutations in the RUNX1 and NRAS genes, and the cytogenetic abnormality, -7/7q-, was demonstrated in children with therapy-related AML. RUNX1 point mutations predominate in those de novo AML and MDS patients with a normal karyotype in leukemic cells. Frequency of RUNX1 point mutations was about 4% in a group of children with de novo AML aged 0-14 years diagnosed during the period of 1998-2009. CONCLUSION: During the course of this investigation, valuable data were obtained concerning RUNX1 gene mutation frequencies in different clinical, morphological, and cytogenetic groups of patients with myeloid malignancies, and its cooperation with other molecular aberrations.