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.

Application of the Moran Model in Estimating Selection Coefficient of Mutated CSF3R Clones in the Evolution of Severe Congenital Neutropenia to Myeloid Neoplasia

“…Let us emphasize that our initial and final fractions of mutant receptor data come from sequencing of samples from patients with SCN. Availability of these sequencing data in papers (Beekman et al, 2012;Skokowa et al, 2014;Klimiankou et al, 2019) is at the stem of our results.…”

“…Changing this assumption leads to different estimates, as it can be tested by modifying the parameter λ in the spreadsheet (λ equals the inverse of the interdivision time). Overall, the estimates span a range from 0 to 0.05, with the exception of patient 13 of publication (Klimiankou et al, 2019) who has a negatively estimated selection coefficient. Figure 3 depicts estimated selection coefficientsŝ from Table 1, plotted against the time interval t 1 − t 0 between the first and the second instance of sequencing.…”

“…One thousand simulations were plotted, with thicker green line denoting their mean, thinner green lines mean ± standard deviation, and black dashed line the solution of Equation (4 AML cases (green circles). Cases labeled as "CN-MDS/AML" in Klimiankou et al (2019) are denoted by black circles.…”

“…An additional effect may be due to the fact that not one, but several types of mutant receptors are observed in MDS. The most frequent is the truncated D715 variant, but there are a number of other, as documented in Beekman et al (2012), Skokowa et al (2014), and Klimiankou et al (2019). Therefore, the basic mutation rate should be multiplied by the number of alternative mutants.…”

Two monogenic disorders masquerading as one: severe congenital neutropenia with monocytosis and non-syndromic sensorineural hearing loss

“…HAX1 variants underlie some autosomal recessive forms and were found to be the underlying cause of SCN in the families originally described by Kostmann [5]. It has been suggested that these variants impair neutrophil maturation via defective CSF3R signaling as the number of G-CSF receptors on myeloid precursors of SCN patients is elevated and majority of SCN patients benefit from the administration of pharmacological doses of granulocyte colony stimulating factor (G-CSF) [6].…”