The Origin and Nature of Tightly Clustered BTG1 Deletions in Precursor B-Cell Acute Lymphoblastic Leukemia Support a Model of Multiclonal Evolution

Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as “drivers” of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL.

影响关键细胞通路的基因发生复发性亚显微缺失，是儿童急性淋巴细胞白血病（pediatric acute lymphoblastic leukemia, ALL）的标志性特征。为深入阐明此类缺失的潜在分子机制，我们针对大型儿童ALL队列中的目标基因之一——B细胞易位基因1（B-cell translocation gene 1, BTG1）的异常发生情况与本质展开了研究。
研究发现，BTG1仅受基因组缺失影响：在722例前B细胞急性淋巴细胞白血病（B-cell precursor ALL, BCP-ALL）患者样本中，65例（9%）检测到此类缺失，而109例T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）样本中均未检出。
本次研究共鉴定出8种不同大小的缺失，所有缺失均聚集于BTG1基因第二个（亦是最后一个）外显子内的端粒侧热点区域，最终导致截短型BTG1通读转录本的表达。
几乎所有缺失区域的两端均存在V(D)J重组信号序列，这强烈提示异常RAG1/RAG2介导的重组是此类缺失的致病机制。
此外，我们在前B细胞急性淋巴细胞白血病细胞的BTG1基因体中检测到高水平的组蛋白H3赖氨酸4三甲基化（histone H3 lysine 4 trimethylation, H3K4me3）——该修饰已知可将RAG酶复合物锚定至DNA——而T-ALL细胞中未检测到此修饰。
BTG1缺失在超二倍体BCP-ALL亚型中极为罕见，但在其他细胞遗传学亚型中占比颇高，包括ETV6-RUNX1阳性与BCR-ABL1阳性BCP-ALL亚型。
通过基于PCR的敏感筛查，我们在前B细胞急性淋巴细胞白血病的多个亚克隆中鉴定出额外的BTG1缺失，其细胞遗传学分布特征一致；在部分病例中，此类亚克隆可在复发时扩增为主要克隆。
综上，本研究结果表明，BTG1缺失可作为特定BCP-ALL亚型白血病发生的“驱动事件”；此类缺失可在易发生异常RAG1/RAG2介导重组的位点，于多个亚克隆中独立产生。本研究结果进一步证实了急性淋巴细胞白血病存在复杂的多克隆进化过程。