Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia
Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia
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DOI：
10.3389/fimmu.2019.00116
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发表时间：
2019-02-28
影响因子：
7.3
通讯作者：
Welte, Karl
中科院分区：
文献类型：
--
作者：
Klimiankou, Maksim;Uenalan, Murat;Welte, Karl
在白血病前期的严重先天性中性粒细胞减少症 (CN) 和明显的急性髓性白血病 (AML) 或骨髓增生异常综合征 (MDS) 患者中，已描述了高频率的获得性 CSF3R（集落刺激因子 3 受体，粒细胞）突变。在这里，我们报告了对 CSF3R 片段的超灵敏深度测序的建立，该片段编码已知在 CN-MDS/AML 患者中发生突变的 G-CSFR 的细胞内“关键区域”。使用该方法，我们实现了 0.01% 的突变等位基因频率 (MAF) 检出率。我们在具有不同遗传背景的 CN 患者中检测到 CSF3R 突变，但在长期接受 G-CSF 治疗的其他类型骨髓衰竭综合征（例如 Shwachman-Diamond 综合征）患者中未检测到 CSF3R 突变。比较来自骨髓和外周血细胞的 DNA 和 cDNA 的 CSF3R 深度测序结果显示，来自外周血多形核中性粒细胞的 cDNA 的灵敏度最高。这种方法能够识别低频 CSF3R 突变克隆，提高灵敏度，并更早检测 CN 患者白血病前 HSC 的致白血病进化过程中获得的 CSF3R 突变。我们建议在诊断时对整个 CSF3R 基因进行测序，以识别患有遗传性功能丧失型 CSF3R 突变的患者，并每年对 CSF3R 关键区域进行超深度测序，以监测 CSF3R 突变的获得。
High frequency of acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a CSF3R segment encoding the intracellular "critical region" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected CSF3R mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of CSF3R deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency CSF3R mutant clones, increases sensitivity, and earlier detection of CSF3R mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function CSF3R mutations and annual ultra-deep sequencing of the critical region of CSF3R to monitor acquisition of CSF3R mutations.