A 2-year-old girl initially presented at the hospital during an episode of recurrent suppurative tonsillitis (9). Physical examination revealed moderately enlarged tonsils but no signs of systemic or severe infection. Her complete blood count (CBC) showed a marked decrease in ANC (0.42 × 10⁹/L [normal range: 2.4–4.8 × 10⁹/L]) and mild anemia (white blood cell [WBC] 4.0 × 10⁹/L [normal range: 6–12 × 10⁹/L]; hemoglobin (Hb) 94 g/L [normal range: 110–160 g/L]; and platelet [PLT] 257 × 10⁹/L [normal range: 100–400 × 10⁹/L]). She had an increased C-reactive protein (CRP) level (170 mg/L [normal range:0–10 mg/L]), procalcitonin (0.14 ng/ml [normal range: 0–0.06 ng/ml]), and erythrocyte sedimentation rate (83 mm/h [normal range: 0–15 mm/h]). Further laboratory tests showed that her serum antistreptolysin O level was normal, and no Epstein–Barr virus DNA was detected in her peripheral blood. Her serum immunoglobulin (IgG, IgM, and IgA) levels were normal. Flow cytometry analysis showed she had a normal peripheral lymphocyte profile (cluster of differentiation 3-positive [CD3⁺], CD19⁺, CD56⁺, CD3⁺CD4⁺, and CD3⁺CD8⁺ cell counts and CD4⁺/CD8⁺ ratio were all in normal ranges for healthy children).

The patient recovered after 1 week of treatment with antibiotics. CBC after her recovery confirmed neutropenia (WBC 5.6 × 10⁹/L, Hb 104 g/L, PLT 349 × 10⁹/L, ANC 0.19 × 10⁹/L, CRP 9.95 mg/L). Cytologic analysis of bone marrow aspirate showed normal cellularity in terms of the proportion of cells in each granulocyte stage and cell morphology. A review of her medical history confirmed that she had low ANC (0.4 × 10⁹/L) starting from 5 months of age but was refractory to recombinant human (rh)G-CSF treatment (15–30 episodes of suppurative tonsillitis between the ages of 1 and 2 years under rhG-CSF treatment). Her parents denied consanguinity and both were healthy with normal ANC.

Whole-exome sequencing of the patient and her parents identified a compound heterozygous mutation in the CSF3R gene (NM_000760.3) in the child, who inherited an intronic mutation (c.64+5G>A) from her father and a frameshift mutation (c.690delC, p.Met231Cysfs^*32) from her mother (Figure 1). The latter was predicted to be damaging by MutationTaster (http://www.mutationtaster.org/). The clinical significance of the intronic mutation (c.64+5G>A) is not clear. Both mutations are present at a very low frequency in the general population (Table 1).

The expression level of CSF3R in neutrophils of the patient and her parents was determined by real-time (rt)PCR and Western blotting. In rtPCR, total RNA extracted from peripherial blood using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was reverse transcribed to cDNA using the PrimeScript RT reagent kit (Takara Bio, Ostu, Japan). The rtPCR was performed on a LightCycler 480 system (Roche, Basel, Switzerland) using the SYBR Premix ExTaq Kit (Takara Bio). The 2^−ΔΔCt method was used to calculate the relative levels of CSF3R mRNA. Primer sequences (Sangong Biotech, Shanghai, China) are 5′-GGGGA GAGAA GCTGG ACTG-3′ and 5′-GTTCA TGAGG CAGGA GAGGT-3′ for CSF3R (product length 578 bp), and 5′-ACATC AAGAA GGTGG TGAAG-3′, and 5′-TGACA AAGTG GTCGT TGAG-3′ for GAPDH (product length 220 bp). For Western blotting, total protein was extracted from neutrophils separated from peripherial blood of the of the patient and her parents using a commercial kit (protein extraction kit [BB-3134], BestBio, Shanghai, China). After determining the protein concentration with the BCA method, whole protein extract was resolved by SDS-PAGE using a 4% acrylamide gel. The proteins were transferred to a PVDF membrane (Millipore, Schwalbach, Germany) that was probed with primary antibodies against CSF3R (ab156878) and GAPDH (ab8245) followed by horseradish peroxidase-conjugated goat antirabbit IgG H&L (ab205718, all from Abcam, Cambridg, UK). Protein bands were visualized by enhanced chemiluminescence using an imaging system (Tanon 6600, Science & Technology Co, Shanghai, China). Quantification of band intensities in Western blotting was performed using ImageJ (version 1.53k) (10). The levels of CSF3R expression in neutrophils between subjects were compared by t-test (R, version 4.0.3, www.r-project.org), and a p < 0.05 is considered statistically significant.

Based on the patient's presentation and the family's genetic profile, SCN type 7 (SCN7, MIM #617014) was diagnosed. After initiating rhG-CSF (5 μg/kg) treatment, the patient's ANC increased from 0.4 to 0.6 × 10⁹/L; however, after rhGM-CSF (5 μg/kg, Topleucon, Amoytop Biotech, Xiamen, China) injection, ANC increased from 0.19 to 1.95 × 10⁹/L (Figure 2). Therefore, the treatment plan was changed to subcutaneous rhGM-CSF (5 μg/kg/day) once ANC was lower than 0.5 × 10⁹/L (on twice a week surveillance). During the next 68 days, the patient received five rhGM-CSF subcutaneous injections but three episodes of suppurative tonsillitis occurred. After observing that overt infection still occurred when ANC was >0.5 × 10⁹/L, we raised the ANC target to 1.0 × 10⁹/L with a decreased dose (3 μg/kg/day) and increased frequency (twice a week) of rhGM-CSF. The patient received eight injections during the next 42 days, and no infection occurred. We then adjusted the injection interval from twice to once a week and monitored ANC once a week before injection and continued to follow up the patient for the next 6 months. There were no other occurrences of infection (Figure 2).

The mRNA and protein levels of CSF3R in perpherial neutrophils were significantly lower in the patient when compared to either her parents or healthy controls (Figure 3). However, parents of the patient showed significantly increased CSF3R mRNA than control subjects but the CSF3R protein level was similar to that of healthy controls (Figure 3).

We searched the literature for the clinical characteristics and treatment of all cases with SCN7 caused by biallelic CSF3R mutation reported since 2014 (Table 2). There are nine pedigrees (13 patients) that have been described to date; six cases were from Turkey (11), nine were female, and all presented repeated infection and required long-term antibiotic treatments. One patient died shortly after birth for unknown reasons (12). The phenotype of SCN7 ranged from severe neutropenia unresponsive to high-dose rhG-CSF treatment (c.998-2A>T and p.W547^* compound heterozygous) (7) to mild neutropenia that does not require active treatment (p.R440^* homozygous) (14). Among the three patients (including patient in current study) who responded to rhGM-CSF treatment, the dose of rhGM-CSF ranged from 3 μg/kg every week to 5 μg/kg every other day (7, 8).