Dataset Information

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

ABSTRACT: 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.

SUBMITTER: Klimiankou M

PROVIDER: S-EPMC6413711 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

Klimiankou Maksim M Uenalan Murat M Kandabarau Siarhei S Nustede Rainer R Steiert Ingeborg I Mellor-Heineke Sabine S Zeidler Cornelia C Skokowa Julia J Welte Karl K

Frontiers in immunology 20190228

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 ...[more]

PMID: 30891028

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Project description: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.

Dataset Information

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

Publications

Ultra-Sensitive <i>CSF3R</i> Deep Sequencing in Patients With Severe Congenital Neutropenia.

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