An activating mutation in the CSF3R gene induces a hereditary chronic neutrophilia

Abstract

We identify an autosomal mutation in the CSF3R gene in a family with a chronic neutrophilia. This T617N mutation energetically favors dimerization of the granulocyte colony-stimulating factor (G-CSF) receptor transmembrane domain, and thus, strongly promotes constitutive activation of the receptor and hypersensitivity to G-CSF for proliferation and differentiation, which ultimately leads to chronic neutrophilia. Mutant hematopoietic stem cells yield a myeloproliferative-like disorder in xenotransplantation and syngenic mouse bone marrow engraftment assays. The survey of 12 affected individuals during three generations indicates that only one patient had a myelodysplastic syndrome. Our data thus indicate that mutations in the CSF3R gene can be responsible for hereditary neutrophilia mimicking a myeloproliferative disorder.

Figure 1.

An inherited mutation in the CSF3R gene in a familial neutrophilia. (A) Pedigree of the family. The black symbols represent affected individuals with neutrophilia and T617N amino acid substitution. The gray symbols represent individuals for whom clinical information was not available. The white symbols represent nonaffected individuals. The genotype is also indicated (N/N, normal subjects; N/M, heterozygous patients). (B) Examples of electrophoregrams from normal subjects or patient 15 DNA (T617N). (C–E) Liquid culture of CD34⁺ cells in the presence of SCF plus G-CSF or SCF (C and D) from controls (n = 5) or patients (n = 2, patients 7 and 15) in which three independent experiments were performed for each patient. Error bars represent means ± SD. *, P < 0.05. (E) Cytological examination at day 21 of culture. Numbers indicate the percentages of mature granulocytic cells. Results are representative of controls (n = 3) or patients (n = 2, patients 7 and 15) in which two independent experiments were performed for each patient. (F) Western blot analysis of P-Jak2, P-STAT3, P-STAT5, P-ERK p42-p44, and P-AKT antibodies in normal or patient CD34⁺ cells stimulated by G-CSF in the presence or not of AZ1480. Results are representative of three independent experiments either with control or patient 15. Black lines indicate that intervening lanes have been spliced out.

Figure 2.

Computational searches of wild-type and T617N G-CSF-R dimers. (A) Molecular structure of the helix dimer of the T617N mutant having the overall lowest energy in the computational searches. The helices have a left-handed crossing angle and an axial separation of 10.5 Å. The interfacial amino acids are highlighted. (B) Plot of the helix interaction energies in the wild-type (black) and T617N (red) TM helix dimers with axial separations of 10.5 Å. In the wild-type G-CSF-R, the dominant interaction is a direct Thr617–Thr617 hydrogen bond between side chain hydroxyl groups. In contrast, there are several strong stabilizing interactions in the mutant. The Asn amide side chain forms hydrogen bonds to the thiol side chain of Cys620. Trp624 forms stabilizing van der Waals contacts with the opposing helix; the indole NH is also able to form an interhelical H bond with Cys620. The total interaction energies were very similar for the lowest energy T617N dimers in computational searches at 10 Å (−80.6 kcal/mol) and 10.5 Å (−81.0 kcal/mol). (C) Cross section of the T617N helix dimer showing hydrogen-bonding interactions involving Thr617 and Cys620. The axial separation is 10.5 Å. (D) Cross section of the T617N helix dimer with an axial separation of 11 Å stabilized by interhelical interactions between Asn617 side chains and between Asn617 and Cys618. The overall interaction energies were generally higher at longer interhelical separations for both the wild-type and mutant dimers, although the T617N dimers were consistently lower in energy than those of the wild-type dimers because of the ability of Asn617 to form interhelical hydrogen bonds.

Figure 3.

CSF3R^T617N mutation yields to MPD in the mouse BM transplant model. BM cells were collected from C57/B6 SJL (CD45.2) mice 2 d after 5-fluorouracil treatment. Lin⁻ cells were purified; cultured for 2 d with IL-3, SCF, IL-6, and TPO; infected twice with the viral particles containing empty vector (Migr), CSF3R^wt, or CSF3R^T617N; and finally injected intravenously into lethally irradiated (825 rad) C57BL6 (CD45.1) mice. After 5 wk, mice were sacrificed and the total number of cells (A, C, and E) or the percentage of cells (B, D, and F) including granulocytes (Gr-1⁺), B lymphocytes (B220⁺), or T lymphocytes (CD3⁺) was measured in CD45.2⁺ donor mouse cells in the blood (A and B), BM (C and D), and spleen (E and F). (G) Spleen from Migr-engrafted (I), CSF3R^wt-engrafted (II), and CSF3R^T617N-engrafted (III) mice. The results are the means ± SD of four mice of each group from two independent experiments. *, P < 0.05 compared with Migr-engrafted mice.

Figure 4.

CSF3R^T617N mutation yields to MPD in the xenotransplantation model. CD34⁺ cells from normal subjects or from patients were intravenously injected into NOG mice and sacrificed at 15 wk. Flow cytometry analysis of human cell engraftment shows the percentage of human CD45⁺ cells (A) and percentage of B (CD19⁺) and myeloid (CD33⁺) cells (B and C) among CD45⁺ cells in the peripheral blood, BM, spleen, and thymus. The results are the means ± SD of four mice for donors and patient from two independent experiments. (D) Peripheral blood progenitors among CD45⁺ cells from donor- or patient-engrafted mice.