Department of Pediatrics, Cancer Biology, and Translational Hematology and Oncology Research, Cleveland Clinic, Cleveland, OH, USA.
Department of Systems Biology and Engineering, Silesian University of Technology, Gliwice, Poland.
Mutagenesis. 2020 Dec 1;35(5):381-389. doi: 10.1093/mutage/geaa027.
Severe congenital neutropenia (SCN) is a rare blood disorder characterised by abnormally low levels of circulating neutrophils. The most common recurrent mutations that cause SCN involve neutrophil elastase (ELANE). The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF), which increases the neutrophil number and improves the survival and quality of life. Long-term survival is however linked to the development of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R (the G-CSF receptor). About 70% of SCN patients with AML harbour additional mutations in RUNX1. We hypothesised that this coding region of CSF3R constitutes a hotspot vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to measure the effect of induced oxidative or ER stress on the mutation rate in our hypothesised hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a green fluorescent protein (GFP) tag were subjected to stress-inducing treatment for 30 days (~51 doubling times). The amplicon-based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all the three genes of interest (partial CSF3R, Csf3r and Runx1), there were more mutations in the GFP region compared with the partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Our data suggest that oxidative or ER stress induction does not promote genomic instability, affecting partial C-terminal of the transduced CSF3R, the endogenous Csf3R and the endogenous Runx1 in Ba/F3 cells that could account for these targets to being mutational hotspots. We conclude that other mechanisms to acquire mutations of CSF3R that help drive the evolution of SCN to MDS/AML.
严重先天性中性粒细胞减少症（SCN）是一种罕见的血液疾病，其特征是循环中性粒细胞水平异常低。导致 SCN 的最常见反复突变涉及中性粒细胞弹性蛋白酶（ELANE）。SCN 的治疗选择是施用粒细胞集落刺激因子（G-CSF），这增加了中性粒细胞数量并改善了生存和生活质量。然而，长期生存与骨髓增生异常综合征/急性髓系白血病（MDS/AML）的发展有关。大约 70%的 MDS/AML 患者获得影响 CSF3R（G-CSF 受体）细胞质结构域的无义突变。大约 70%的患有 AML 的 SCN 患者在 RUNX1 中存在额外的突变。我们假设 CSF3R 的这个编码区构成了一个易受突变的热点，这些突变是由过度氧化应激或内质网（ER）应激引起的。我们使用小鼠 Ba/F3 细胞系来测量诱导的氧化或 ER 应激对我们假设的外源性人 CSF3R 热点、内源性 Csf3r 和 Runx1 中相应区域的突变率的影响。用与人 CSF3R 的 C 末端部分融合的 GFP 标签的 cDNA 转导的 Ba/F3 细胞接受应激诱导处理 30 天（约 51 次倍增）。第 15 天和第 30 天样本的基于扩增子的靶向深度测序数据表明，尽管在所有三个感兴趣的基因（部分 CSF3R、Csf3r 和 Runx1）中都观察到了增加的突变，但与部分 CSF3R 区域相比，GFP 区域的突变更多。我们的研究结果还表明，在 Ba/F3 细胞中，诱导应激的化学处理与突变之间没有相关性。我们的数据表明，氧化或 ER 应激诱导不会促进基因组不稳定性，影响转导的 CSF3R、内源性 Csf3r 和内源性 Runx1 的 C 末端部分，这些靶标可能成为突变热点。我们得出结论，其他机制可获得 CSF3R 的突变，有助于 SCN 向 MDS/AML 的进化。
