Efficacy of Low-Dose rhGM-CSF Treatment in a Patient With Severe Congenital Neutropenia Due to CSF3R Deficiency: Case Report of a Novel Biallelic CSF3R Mutation and Literature Review

This study reports the clinical manifestations, genetics, and efficacy of treatment with the efficacy of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-GSF) of a 2-year-old female patient with severe congenital neutropenia (SCN) type 7 (SCN7) caused by novel biallelic mutations in the colony-stimulating factor 3 receptor (CSF3R) gene. Genetic diagnosis of the patient was performed by whole-exome and Sanger sequencing. Expression of the CSF3R gene in the peripheral neutrophils of the patient was detected by real-time PCR and Western blotting. The patient presented with recurrent suppurative tonsillitis and decreased absolute neutrophil count <0.5 × 109/L. Novel heterozygous mutations were found to be inherited from each parent (maternal c.690delC [p.met231Cysfs*32] and paternal c.64+5G>A). The patient's neutrophils had lower CSF3R mRNA and protein levels than those of the parents. Low-dose rhGM-CSF (3 μg/kg/day once a week) prevented recurrent infection in the patient. These results demonstrate that the clinical manifestations of SCN7 with biallelic CSF3R mutations and downregulated CSF3R can be effectively treated with rhGM-CSF.

The patient recovered after 1 week of treatment with antibiotics. CBC after her recovery confirmed neutropenia (WBC 5.6 × 10 9 /L, Hb 104 g/L, PLT 349 × 10 9 /L, ANC 0.19 × 10 9 /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 × 10 9 /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 − Ct 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.

Diagnosis and Treatment
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 × 10 9 /L; however, after rhGM-CSF (5 µg/kg, Topleucon, Amoytop Biotech, Xiamen, China) injection, ANC increased from 0.19 to 1.95 × 10 9 /L (Figure 2). Therefore, the treatment plan was changed to subcutaneous rhGM-CSF (5 µg/kg/day) once ANC was lower than 0.5 × 10 9 /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 × 10 9 /L, we raised the ANC target to 1.0 × 10 9 /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 Expression of CSF3R in Peripherial Neutrophils of the Patient
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).

Literature Review
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).

DISCUSSION
In this report, we describe the case of a 2-year old girl with recurrent infection and neutropenia who was genetically diagnosed with SCN7 caused by biallelic inactivating mutations in CSF3R. Similar to most previously reported cases, our patient did not respond to rhG-CSF, but rhGM-CSF treatment showed satisfactory efficacy and was titrated to a lower dose (3 µg/kg/day once a week). SCN is a group of rare congenital hematologic diseases caused by germline mutations with an incidence rate of ∼1/200,000 (15). To date, more than 20 SCN-related genes have been identified, including ELANE (16), GFI-1 (17), HAX1 (18), G6PC3 (19), VPS45 (20), JAGN1 (21), and CSF3R (7,22). The mortality of SCN is attributable to an increased risk of severe bacterial infection (23). Since 1987, the use of rhG-CSF has significantly improved the prognosis and quality of life of patients with SCN (24,25). In most patients, the frequency of bacterial infection is significantly reduced when ANC is maintained above 1.0 × 10 9 /L (26).
G-CSF and its receptor are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice, and zebrafish (27)(28)(29). The ineffectiveness of G-CSF in patients with SCN7, which is caused by biallelic loss-of-function in the G-CSF receptor gene (CSF3R) at a high dose (up to 110 µg/kg/day) (7), demonstrates the importance of interaction between G-CSF and its receptor for the maintenance of normal neutrophil numbers.
CSF3R dimerizes to form a functional receptor (7). A previous report shows that patients with bialliac CSF3R  deficiency can present mild neutropenia that does not require active treatment (14) and suggests that other compensatory mechanisms are involved in the maintenance of adequate number of neutrophils (30). Our results show that CSF3R mRNA expression was decreased in the patient but was increased in both parents who were heterozygous carriers; on the other hand, Western blot analysis showed that the parents had normal CSF3R protein levels. This is similar to previous findings that the expression of nonfunctional CSF3R was increased in patients with biallelic CSF3R mutations (12), and suggests that a genetic compensation mechanism rather than non-sensemediated decay was activated to maintain the normal CSF3R levels (31,32).
Both G-CSF and GM-CSF play important roles in the differentiation and development of myeloid cells. The responsiveness to rhGM-CSF of patients with CSF3R mutations can be attributed to granulocyte stimulation by GM-CSF and the activation of CSF2R (30,33,34). GM-CSF sequentially induces myeloid proliferation, differentiation, and maturation (35) with mature neutrophils released into the peripheral circulation after about 5-8 days (36). We believe that rhGM-CSF (3 µg/kg/day) once a week can be effective for maintaince when the patient is free from overt infection (i.e., no additional neutrophil consumption). However, the current cases are not sufficient to summarize a clear relation between genotype and responsiveness to rhGM-CSF, an individualized treatment plan based on the severity of infection, mutation type, and response to treatment is probably needed for each patient. The current literature did not confirm whether a patient with SCN7 can be unresponsive to either rhG-CSF or rhGM-CSF, and among those patients, hematopoietic stem cell transplantation can serve as a treatment option (37,38).

CONCLUSIONS
In summary, our study shows that patients with SCN7 caused by biallellic CSF3R mutations with decreased CSF3R expression respond well to adequate rhGM-CSF treatment. Low-dose GM-CSF once weekly is capable of maintaining our patient free from recurrent infection.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Children's Hospital of Fudan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.