Lopes Bruno Almeida, Meyer Claus, Barbosa Thayana Conceição, Zur Stadt Udo, Horstmann Martin, Venn Nicola C, Heatley Susan, White Deborah L, Sutton Rosemary, Pombo-de-Oliveira Maria S, Marschalek Rolf, Emerenciano Mariana
Pediatric Hematology-Oncology Program, Research Center, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil.
Diagnostic Center of Acute Leukemia/Institute of Pharmaceutical Biology/ZAFES, Goethe-University of Frankfurt, Biocenter, Germany.
Oncotarget. 2016 Aug 16;7(33):53064-53073. doi: 10.18632/oncotarget.10590.
IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in childhood B-cell precursor acute lymphoblastic leukemia. Because of its clinical importance, we previously mapped breakpoints of intragenic deletions and developed a multiplex PCR assay to detect recurrent intragenic ΔIKZF1. Since the multiplex PCR was not able to detect complete deletions (IKZF1 Δ1-8), which account for ~30% of all ΔIKZF1, we aimed at investigating the genomic scenery of IKZF1 Δ1-8. Six samples of cases with IKZF1 Δ1-8 were analyzed by microarray assay, which identified monosomy 7, isochromosome 7q, and large interstitial deletions presenting breakpoints within COBL gene. Then, we established a multiplex ligation-probe amplification (MLPA) assay and screened copy number alterations within chromosome 7 in 43 diagnostic samples with IKZF1 Δ1-8. Our results revealed that monosomy and large interstitial deletions within chromosome 7 are the main causes of IKZF1 Δ1-8. Detailed analysis using long distance inverse PCR showed that six patients (16%) had large interstitial deletions starting within intronic regions of COBL at diagnosis, which is ~611 Kb downstream of IKZF1, suggesting that COBL is a hotspot for ΔIKZF1. We also investigated a series of 25 intragenic deletions (Δ2-8, Δ3-8 or Δ4-8) and 24 relapsed samples, and found one IKZF1-COBL tail-to-tail fusion, thus supporting that COBL is a novel hotspot for ΔIKZF1. Finally, using RIC score methodology, we show that breakpoint sequences of IKZF1 Δ1-8 are not analog to RAG-recognition sites, suggesting a different mechanism of error promotion than that suggested for intragenic ΔIKZF1.
IKZF1缺失（ΔIKZF1）是儿童B细胞前体急性淋巴细胞白血病复发的重要预测指标。鉴于其临床重要性，我们之前绘制了基因内缺失的断点，并开发了一种多重PCR检测方法来检测复发性基因内ΔIKZF1。由于多重PCR无法检测到占所有ΔIKZF1约30%的完全缺失（IKZF1 Δ1-8），我们旨在研究IKZF1 Δ1-8的基因组情况。通过微阵列分析对6例IKZF1 Δ1-8病例的样本进行了分析，该分析鉴定出7号染色体单体、7号等臂染色体以及在COBL基因内出现断点的大片段间质缺失。然后，我们建立了多重连接探针扩增（MLPA）检测方法，并在43例诊断为IKZF1 Δ1-8的样本中筛选7号染色体内的拷贝数改变。我们的结果显示，7号染色体内的单体和大片段间质缺失是IKZF1 Δ1-8的主要原因。使用长距离反向PCR进行的详细分析表明，6例患者（16%）在诊断时存在从IKZF1下游约611 Kb的COBL内含子区域开始的大片段间质缺失，这表明COBL是ΔIKZF1的一个热点区域。我们还研究了一系列25例基因内缺失（Δ2-8、Δ3-8或Δ4-8）和24例复发样本，发现了一个IKZF1-COBL尾对尾融合，从而支持COBL是ΔIKZF1的一个新热点区域。最后，使用RIC评分方法，我们表明IKZF1 Δ1-8的断点序列与RAG识别位点不同，这表明与基因内ΔIKZF1所提示的错误促进机制不同。
