PMC8296982_01
Neutrophil counts before and after granulocyte colony-stimulating factor (G-CSF) treatment. (A) Neutrophil count in patient 1 before G-CSF treatment. (B) Neutrophil count during G-CSF treatment in patient 1; Measurements after discontinuation of G-CSF treatment are shown on the right side of the dotted line. (C) Neutrophil count before G-CSF treatment in patient 2. (D) Neutrophil count during G-CSF treatment in patient 2. (E) Images of peripheral blood samples from patient 2 before and during G-CSF treatment, stained with Wright's stain. (F) Neutrophils dominate Ficoll-Paque enriched SCN4 granulocyte fractions. Flow cytometry analysis was performed on Ficoll-Paque enriched granulocytes from patient 2 and healthy control to examine the number of neutrophils. The percentages of myeloid cells (CD11b+CD45+CD16+) and neutrophils (CD11b+CD45+CD16+) were determined.
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Laboratory data revealed baseline absolute neutrophil counts (ANC) < 1 x 109/L, which were noncyclic and transiently reversible by infection in both patients before G-CSF treatment ( Figures 1A, C ), and > 1 x 109/L following G-CSF treatment ( Figures 1B, D ). ANC has remained > 1 x 109/L for 6 months following G-CSF treatment suspension in patient 1 ( Figure 1B ). Hematoxylin and eosin staining and microscopic examination of blood slides from patient 2 during initial G-CSF treatment showed that peripheral blood granulocytes gradually changed from mononuclear to multinucleated, and granulocyte count stabilized ( Figure 1E ). The results suggested that, unlike SCN1 patients, neutrophils are the dominating granulocytes in the blood of SCN4 patients ( Figure 1F ).
Deletions and point mutations detected by targeted deep sequencing. (A) Schematic representation of G6PC3 gene mutations in the two patients. (B) Sequence analysis of G6PC3 in patient 1, his father, and his mother. (C) Sequence analysis of G6PC3 in patient 2, his father, and his mother. ClustalW alignment of human, chimpanzee, macaque, dog, cow, mouse, and rat G6PC3 protein sequences and mutations detected in patients 1 (D, E) and 2 (F, G). (D) amino acids 85-111, the conserved glutamine at position 99 is highlighted in a black box; (E) amino acids 235-261, the conserved methionine at position 256 is highlighted in a black box; (F) amino acids 235-261, the conserved arginine at position 253 is highlighted in a black box; (G) amino acids 180-206, the conserved tyrosine at position 199 is highlighted in a black box.
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Mutations were detected in both patients and their family members ( Table 4 ,  Figures 2A-C ). Our group has reported the mutations in Patient 1 previously, which were the heterozygous mutations in exons 2 and 6: a heterozygous missense mutation (c.295C>T, p.Q99X) in exon 2 inherited from his father, and a heterozygous deletion (c.766_768del, p.256_256del) in exon 6 inherited from his mother ( Figure 2B ), Patient 2 had heterozygous mutations in exons 5 and 6: his father carried a heterozygous missense mutation (c.758G>A, p.R253H) in exon 6, and his mother carried a heterozygous missense mutation (c.596A>G, p. Y199C) in exon 5 ( Figure 2C ). SNP databases and were not observed in 1000 control subjects. Separating intolerant from tolerant (SIFT) analysis shows that these residues are highly conserved among species ( Figures 2D-G ).
CXCR4 expression on neutrophils and NK cells. (A) (Left) Representative histograms of neutrophils from patient 2 during G-CSF treatment and healthy controls. (Right) The mean fluorescence intensity (MFI) of CXCR4 in neutrophils from patient 2 and healthy controls (n=3). (B) Representative histograms of NK cells from patient 1 during and after discontinuation of G-CSF treatment (Left), patient 2 before and during G-CSF treatment (Right), and healthy controls. (C) The MFI of CXCR4 on NK cells from patient 1 after discontinuation of G-CSF treatment, patient 2 before and during G-CSF treatment and healthy controls (n=3).
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The CXCR4 expression on neutrophils from patient 2 (MFI = 910) was higher during G-CSF treatment than in the HC samples (MFI = 299 +- 101.9) ( Figure 3A ). Similarly, the MFI of CXCR4 expression on CD56+NK cells was also increased in patient 2 before he received G-CSF treatment (MFI =411), and almost returned to normal levels after G-CSF treatment (MFI =112) ( Figures 3B, C ).
Detection of respiratory burst activity induced by PMA stimulation. (A) Whole blood was incubated in the absence or presence of PMA, as indicated. Respiratory burst was detected via conversion of DHR-123 in neutrophils. Median fluorescence intensity of the entire neutrophil-gated cell population, enclosed by the marker bar, is specified in each plot. (B) Mean (+- SD) respiratory burst stimulation index (SI) values in healthy controls and patients 1 and 2.
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The respiratory burst capacity of stimulated neutrophils in both patients was low, and ROS generation and release in response to PMA were clearly diminished in patient 1 after discontinuation of G-CSF treatment and in patient 2 during G-CSF therapy, compared with HC samples ( Figure 4A ). The SI values in patients 1 and 2 were 13.31 and 11.92, respectively, which were significantly lower than those in HC samples (SI = 105.4 +- 23.42) ( Figure 4B ).
Apoptosis of human neutrophils analyzed using annexin V and propidium iodide staining. (A) Neutrophils from healthy controls (HCs) and patient 1 following cessation of granulocyte colony-stimulating factor (G-CSF) treatment, and patient 2 before and during G-CSF treatment, at baseline and following culture with or without lipopolysaccharide (LPS) or FAS for 16 h. Cells were stained for annexin V and propidium iodide, analyzed using flow cytometry. Mean (+- SD) percentages of live neutrophils (B), total apoptotic neutrophils (C), and late apoptotic neutrophils (D) among total neutrophils at baseline. Mean (+- SD) percentages of live neutrophils (E), total apoptotic neutrophils (F), and late apoptotic neutrophils (G) among total neutrophils after culture without stimulation for 16 h. Mean (+- SD) percentages of live neutrophils (H), total apoptotic neutrophils (I), and late apoptotic neutrophils (J) among total neutrophils after culture with LPS for 16 h. Mean (+- SD) percentages of live neutrophils (K), total apoptotic neutrophils (L), and late apoptotic neutrophils (M) among total neutrophils after culture with FAS for 16 h.
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In addition, we analyzed the percentages of apoptotic cells following incubation with LPS or FAS for 16 h, to determine apoptosis rates in activated neutrophils and neutrophils after induction of apoptosis, respectively ( Figure 5A ). By contrast, the percentage of apoptotic cells and late apoptotic cells in patients 1 and 2 before G-CSF treatment were significantly higher than those in HCs at baseline, while those in patient 2 after treatment were similar to HCs ( Figures 5B-D ). FAS for 16 h, the percentages of live cells were lower, and those of total apoptotic cells higher, in patients 1 and 2 before and during G-CSF treatment, than those in HCs. Furthermore, following stimulation with LPS, the percentages of late apoptotic cells were slightly higher in patients 1 and 2 during G-CSF treatment and significantly higher in patient 2 before G-CSF treatment ( Figures 5H-J ). Nevertheless, following FAS stimulation, the percentage of late apoptotic cells was significantly higher in patient 1, while in patient 2 during G-CSF treatment, the percentage was only slightly higher ( Figures 5K-M ).
Expulsion of NETs from neutrophils. (A) Fluorescence generated by neutrophils from patient 1 and healthy controls at each time-point following incubation in the presence or absence of PMA stimulation. (B) Fluorescence generated by neutrophils from patient 2 and healthy controls at each time-point following incubation in the presence or absence of PMA stimulation.
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Analysis of neutrophils from both patients during G-CSF therapy and HC using this system demonstrated that NETs formation was significantly defective in neutrophils from both patients ( Figures 6A, B ).