. These computational data strongly support the notion that Asn617 stabilizes an active dimeric orientation for G CSF R by forming interhelical hydrogen bonds. To determine the impact of the mutant G CSF RT617N on G CSF AZD7762 sensitivity, we measured granulocytic proliferation and differentiation from CD34 cells in in vitro experiments. In the presence of stem cell factor and G CSF, proliferation was not significantly different between normal subjects and affected patients. In contrast, SCF alone induced survival of control CD34 cells, whereas it elicited proliferation on patient cells. We also observed a hypersensitivity to G CSF of patient CD34 cells that was confirmed by clonogenic assays in methylcellulose.
Moreover, when CD34 cells from normal subjects and affected individuals were grown in the presence of G CSF, cells differentiated into mature granulocytes, as indicated by cytological examination. In contrast, in the presence of SCF alone, only cells from patients acquired the terminal granulocytic CD11b antigen and matured into neutrophilic BMS-540215 polymorphs. Collectively, these results show that mutant G CSF RT617N induces a G CSF independent granulocytic proliferation and differentiation. cells/mm3. In the peripheral blood, a 3 to 20 fold increase in the percentage of circulating CD34 cells was observed. The BM of two analyzed affected individuals contained an increase in granulocyte precursors without an excess of blasts. The karyotype was normal, Bcr Abl transcripts and JAK2V617F were not detected. All affected patients except patient 15 had no clinical symptoms.
Based on the autosomal dominant pattern of inheritance of the disorder and the high level of blood CD34 cells, we tested the hypothesis that neutrophilia in this family resulted from activation of the G CSF R signaling. Because G CSF concentration in the serum was below the detection limit, we sequenced the CSF3R gene. We found a heterozygous C to A substitution at nucleotide 2,088 that leads to a threonine to asparagine substitution. This heterozygous point mutation was observed in the 12 affected individuals but not in the 4 healthy family members, and was segregated with the neutrophilia. Moreover, the mutation was found in all generations and in as many affected men as women. Finally, the mutation was transmitted with an autosomal dominant pattern of inheritance with complete penetrance.
The CSF3RT617N mutation has already been described as an activating mutation found in 2 out of 555 patients with acute myeloid leukemia. In these two cases, the mutation was acquired because it disappeared after complete remission achievement and was not detected at relapse. This last result demonstrates that the CSF3RT617N mutation was a secondary event in the leukemic process. We studied whether this mutation could be found in sporadic cases of unexplained neutrophilia and investigated 40 cases by allele specific PCR, but we did not find any other positive case suggesting that this activating mutation is rare. The G CSF RT617N mutation is located in the transmembrane domain of the receptor, analogously to the MPLS505N the empty vector, and engrafted the infected cells into irradiated hosts. Mice transplanted with cells expressing the mutant G CSF RT617N developed neutroph