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.

Keywords: G-CSFR mutations; deep-sequencing; leukemogenesis; pre-leukemia; severe congenital neutropenia.

Figure 1

Distribution of acquired mutations in exon 17 of CSF3R in CN and CN-MDS/AML patients. Schematic representation of exon 17 of the CSF3R gene along with protein sequence (NP_000751.3) showing four conserved tyrosine residues. The amino acid positions corresponding to exon 17 are indicated. The intracellular critical region of CSF3R is marked in red. Amino acid (AA) positions of the CSF3R gene mutations are confined to the region between amino acid residues 715 and 787. Mutations in G-CSFR associated with leukemia and MDS in CN are denoted by “AML” or “pre-B ALL”, ^*- stop codon. (B) Inter-run reproducibility between CSF3R DNA deep sequencing runs. Inter-run reproducibility was assessed by sequencing of 13 CSF3R mutations from 11 CN patients in two separate runs.

Figure 2

(A) Comparison of CSF3R mutant allele frequencies in CN and genetically unclassified CN patients. No significant difference in MAF of CSF3R mutations between CN (n = 163) and genetically unclassified CN (n = 48) groups was observed; n.s.- not significant. (B) The acquisition of CSF3R mutations is not associated with higher rhG-CSF doses. Patients were divided into 2 groups based on the presence (n = 40) or absence (n = 54) of CSF3R mutations detected by cDNA deep-sequencing. rhG-CSF doses at the time of sample collection are plotted for the both groups; n.s., not significant.

Figure 3

Comparison of deep sequencing results in different types of peripheral blood (PB) and bone marrow (BM) samples of CN and CyN patients. Frequency of CSF3R mutant clones in different types of PB and BM samples from ELANE-CN patients (A,B), CyN patient (C) and HAX1-CN patients (D,E) was analyzed using cDNA deep sequencing, as described in Material and Methods section. The CSF3R mutant clones are indicated based on the relative amino acid positions of mutations.

Figure 4

Time-course of CSF3R mutations in sequential samples from CN patients detected by cDNA and DNA deep sequencing. (A–D) The percentage of cells expressing mutant CSF3R at different time points (in years), starting from the date of the first CSF3R mutation analysis in a given patient, is plotted. All mutations are considered to be heterozygous. The CSF3R mutant clones are indicated based on the relative amino acid positions of mutation sites. The number of cells with CSF3R mutation is estimated to be twice the number of reads supporting a mutant allele, ^*- stop codon. (E) Time-course of CSF3R mutations occurrence and frequency of mutant alleles in DNA samples from CN patients detected by CSF3R DNA deep sequencing. The frequency of mutant alleles at different time points (in years) in a given patient is plotted. The number of cells with CSF3R mutation is estimated to be twice the number of reads supporting a mutant allele, ^*- stop codon.