Análise genômica de deleções completas de IKZF1 em leucemias linfoblásticas agudas pediátricas / [Genomic analysis of complete IKZF1 deletions in pediatric acute lymphoblastic leukemias]

Introdução: A leucemia de células precursoras B (LLA-CPB) pediátrica é caracterizada por alterações citogenético-moleculares recorrentes, cuja identificação é essencial para aestratificação de risco terapêutico e adequação do tratamento ao risco de recaída em cada paciente. Recentemente estudos genômicos identificaram que deleções em IKZF1 (ΔIKZF1)podem ser preditivas de maior risco de recaída. As ΔIKZF1 foram rastreadas inicialmente por amplificação multiplex dependente de ligação de sondas (MLPA), no entanto, ametodologia é pouco sensível para a avaliação de doença residual mínima. Para tanto, estudos recentes desenvolveram técnicas de PCR multiplex (MP-PCR). Como as MP-PCRnão detectam IKZF1 Δ1-8 (~30% das LLA-CPB pediátricas), este trabalho teve como objetivo investigar as características das IKZF1 Δ1-8 nas LLA-CPB pediátricas. Metodologia: Os sujeitos desta pesquisa foram crianças e adolescentes (< 18 anos) comLLA-CPB e IKZF1 Δ1-8. Inicialmente uma coorte de descoberta (n = 6) foi caracterizada por microarranjo. Em seguida, alterações de número de cópias (CNAs) no cromossomo 7 foramavaliadas na coorte de investigação (n = 45) através de MLPA customizado. A partir dos dados de CNA, os pontos de quebra das deleções foram sequenciados por PCR multiplexou PCR invertida de longa distância. Com as sequências de ponto de quebra, investigamos sequências sinais de recombinação de RAG (RSSs), além de motivos CpG, estrutura secundária do DNA nas regiões de quebra e dados públicos do ENCODE de DNase eChIP-seq. Características demográficas e CNAs de amostras com IKZF1 Δ1-8 e demais deleções foram comparadas no programa SPSS 18, enquanto diferenças na distribuição de motivos nas regiões de quebra foram avaliados no GraphPad Prism 5. Valores p < 0,05 foram interpretados como estatisticamente significantes...

Background: Childhood B-cell precursor acute leukemia is characterized by recurrent cytogenetic and molecular alterations. Their identification is crucial for both risk stratificationand treatment management of patients. Recently, genomic studies have identified IKZF1 deletions (ΔIKZF1) as a valuable predictor of relapse. ΔIKZF1 have been screened by multiplex probe amplification assay (MLPA) in various studies, although the methodologylacks sensitivity for minimal residual disease evaluation. Therefore, novel studies have developed multiplex PCR (MP-PCR) tests with greater sensitivity. Since MP-PCR is still unable to identify IKZF1 Δ1-8 (~30% of recurrent deletions of BCP-ALL), this study aimed to investigate the main characteristics of childhood BCP-ALL cases with IKZF1 Δ1-8. Methods: This study enrolled children aged <18 years diagnosed with BCP-ALL and IKZF1 Δ1-8. First,a discovery cohort (n = 6) was analyzed with CytoScan HD array. Thereafter, CNAs within chromosome 7 were alalyzed in a validation cohort (n = 45) using an in-house MLPA. Thebreakpoints were sequenced after multiplex (MP-PCR) and long-distance inverse PCR (LDIPCR). After all, the possible mechanisms underlying occurrence of deletions were alsoinvestigated using a nucleotide-based similarity approach, along with DNase and ChIP-seq data retrieved from ENCODE database. Statistical analysis of demographic characteristics were compared with SPSS 18, while sequence motifs analyzed on GraphPad Prismsoftware...

Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination.</p><p><b>METHODS</b>TCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique.</p><p><b>RESULTS</b>RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation.</p><p><b>CONCLUSIONS</b>RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.</p>

Expression of human recombinase activating gene 1 and recombinase activating gene 2 [rag 1 and rag 2] in acute lymphoblastic leukemia

The process of V[D]J recombination is limited and controlled by the enzymatic activity of cytoplasmic proteins called recombinase which are the products of the activating two genes called Recombinase Activating Gene 1 and Recombinase Activating Gene 2 [RAG1 and RAG 2]. Both genes are expressed in immature B and T lymphocytes and activated V[D]J rearrangement in Ig and TCR genes which are directed in cis by recombination signals sequences [RSSs]. Also, they show variable expression in lymphoid malignancies of both B and T-cell types. This study aimed to focus on the role of the two cytoplasmic proteins [RAG1 and RAG2] in the developing of both B and T ALL, correlation to the stages of differentiation and also, their possible prognostic significance. This study included 40 newly diagnosed acute lymphoblastic leukemic patients [ALL] their age ranged from 1-10 years. Assessment of RAG1 and RAG2 expression by reverse transcriptase polymerase chain reaction [RT-PCR] was done using peripheral blood sample. RAG1 and RAG2 positive expression was higher in frequency in pediatric ALL cases compared to control group, the difference was statistically significant [P<0.01]. Among the RAG1 and RAG2 positive ALL cases 25% were pro-B-ALL and 25% CALL and 31.2% pre-B-ALL and 18.8% were early T-ALL. On the other hand RAG1 and RAG2 negative ALL cases showed higher frequency of CALL phenotype [83.3%] while pro-B ALL, pre-B ALL and early-T ALL were 4.2% and 8.3% and 4.2% respectively. The RAG1 and RAG2 initially positive ALL cases studied had poor prognosis, where 37.5% relapsed and 25%, while 37.5 were in continuous complete remission. However among the RAG1 and RAG2 negative ALL cases studied, 91.7% had good prognosis with complete remission, while only one patient [4.2%] relapsed and one died [4.2%]. A statistical significant association between RAG1 and RAG 2 positive expression and poor prognosis was noticed. RAG1 and RAG2 could be used as prognostic marker in lymphoid malignancies and its sub-classification