This is a confirmed case of CN in a Chinese infant with the c.170 C > A variant in the ELANE gene. The child has presented with various degrees of neutropenia since the neonatal period, accompanied by recurrent infections. A bone marrow examination revealed maturation disorders in neutrophils, and elevated percentages of monocytes and eosinophils, along with an increased peripheral monocyte and platelet count, all of which support the diagnosis of CN in this patient [4, 11]. Multiple online prediction analyses and ACMG criteria support the pathogenicity of this de novo missense variant [12, 13]. During the early neonatal stage, the patient consistently exhibited severe neutropenia (ANC < 0.5 × 10^9/L). Periodic fluctuations in neutrophil counts observed twice a week during particular months suggest a cyclical pattern. Therefore, the diagnosis of our patient is appropriate for the intermediate phenotype of CN [8]. To our knowledge, this is the first reported case of CN associated with the rare pathogenic variant, c.170 C > A variant in the ELANE gene in the Chinese mainland and a rare variant globally.

CN exhibits genetic heterogeneity and can be inherited through autosomal dominant, autosomal recessive, and X-linked inheritance [11]. More than half of CN cases are linked to variants in the ELANE gene, which contains five exons and is located on human chromosome 19 [11]. Although the mechanisms underlying CN due to ELANE variants are not yet fully understood, there are two hypotheses [14]. On one hand, ELANE variants lead to the mislocalization of NE. The variants in the terminal exons can affect the spatial structure and biological function of the carboxyl-terminal (C-terminal) protein and peptide. Variants in exons 4 and 5 have been shown to typically impact the amino acid composition at the C-terminus, disrupting the disulfide bond domain and causing the loss of the binding site for the lysosomal adaptor protein 3 (AP3). This results in the mislocalization of NE to the cell membrane, inducing premature apoptosis, impaired granulocytic differentiation, and degradation of G-CSF and its receptor, ultimately leading to CN [6, 15, 16]. On the other hand, ELANE variants disrupt protein folding, leading to the accumulation of misfolded proteins. This excessive accumulation induces endoplasmic reticulum (ER) oxidative stress and the unfolded protein response (UPR), affecting cell apoptosis and granulocytes [17, 18]. The Ala57 residue in the ELANE gene is a common mutational site. According to protein structure prediction, c.170 C > A will result in the replacement of alanine (Ala) with aspartic acid (Asp) at the Ala57 residue. The carboxyl terminus (C terminus) of Asp affects the spatial structure and biological function of proteins and peptides, destroys the transmembrane domain and causes the mislocalization of neutrophil elastase molecules [14, 16, 19]. Ala is a non-polar aliphatic amino acid with hydrophobicity, while Asp is an acidic amino acid with hydrophilicity. Asp increases the hydrogen bond binding of adjacent amino acids, which may affect the stability of the protein and prevent proper folding, thereby triggering the unfolded protein response [14, 18, 20].

The relationship between ELANE variants and clinical phenotypes, as well as the natural course of the disease, is complicated [11, 21, 22]. ELANE-related CN can manifest as Severe CN (SCN) and CyN. Generally, SCN was diagnosed at a younger age, typically under 1 year, while approximately 50% of CyN patients were diagnosed after 18 months [22]. In terms of CBC, the ANC in SCN is usually less than 0.5 × 10^9/L, while CyN exhibits periodic fluctuations approaching 2.0 × 10^9/L. Additionally, the peripheral monocyte count is typically significantly elevated in SCN. A clinical study in France indicated that SCN patients with ELANE variants had a median monocyte count of 1.3 × 10^9/L while it was in the normal range among CyN patients [22]. The bone marrow cytology examination suggests that the percentage of either promyelocytes or metamyelocytes in ELANE-associated SCN was less than 10% [22]. As for the treatment, SCN typically requires higher doses and more frequent G-CSF injections. Compared to CyN, SCN patients are more prone to bacterial infections and hematological malignancies [2, 22]. It has been reported that the incidence of AML/MDS in ELANE-associated SCN was over 16% [2], and another study demonstrated that the cumulative incidence of leukemia associated with ELANE-related SCN patients reached 21% during a median follow-up of 8 to 9 years [22].

The classic phenotypes of SCN and CyN are easily distinguishable, but some phenotypes in CN present diagnostic challenges due to the variability in the severity of neutropenia and differing clinical histories and hematologic features. Periodic oscillations in neutrophil counts can be observed in both SCN and CyN patients, along with the different durations of neutropenic periods in CyN, further complicating diagnosis. The relationship between specific ELANE gene variants and their corresponding phenotypes may be helpful in making a differentiation. Previous studies have indicated that variants in CyN and SCN often differ, with intronic variants in the ELANE gene typically associated with CyN, while exonic variants are more often diagnosed as SCN [2, 23]. Patients with SCN are more prone to progressing to MDS/AML. A retrospective study involving data from the Severe Chronic Neutropenia International Registry (SCNIR) with ELANE variants showed that during the follow-up period of up to 27 years, sequence variants of exon 2 were more associated with SCN, with only 31% of patients (18/58) diagnosed as CyN. Overlapping variants existed in CyN and SCN patients, where the same variant can lead to both CyN and SCN, such as F43L (F, phenylalani ne; L, leucine) and A61V (A, Alanine; V, valine) located in exon 2, etc. Other variants in exon 2, such as A57T (T, threonine) and A57V, were specific to SCN. The incidence of pneumonia in patients with SCN-specific variants was 63%, while it was 19% in patients with CyN-specific variants and 42% in patients with overlapping variants [2]. To further differentiate ELANE-related SCN and CyN, a study analyzed the interpretation of sequence variants on mRNA expression, protein expression, and activity in peripheral blood and bone marrow. The results showed significantly elevated ELANE mRNA expression in granulocytes of SCN and CyN patients, with markedly reduced NE activity relative to healthy individuals [23]. The patients with SCN had lower relative NE activity compared to those with CyN, but due to limited samples, the threshold for distinguishing relative NE activity between SCN and CyN remained uncertain, which may be a direction for future work [23].

As for the phenotypes of the Ala57 residue in exon 2, there are some differences in the natural course of the disease among patients. The c.170 C > A variant in the ELANE gene was identified in our patient, previously reported in a man with CyN [20]. The patient experienced self-limiting periodic fever episodes and periodic neutropenia since the age of 7, with a definitive diagnosis made at the age of 20 [20]. Although the variant (p.Ala57Asp) has not yet been reported in SCN patients, previous reports have documented pathogenic single nucleotide variants affecting the same codon leading to SCN (Ala57Thr/Val; Thr,threonine; Val, valine) [19, 24,25,26]. A Korean boy was diagnosed with SCN with the variant c.170 C > T (p.Ala57Val) at 5 years old. He had recurrent skin, ear, and respiratory infections, ANC < 0.2 × 10^9/L, and a poor response to G-CSF therapy, with persistent neutropenia and recurrent infections despite increasing the daily dose by 20 µg/kg [19]. After receiving hematopoietic stem cell transplantation (HSCT) from his healthy brother, the ANC returned to the normal range 13 months later. In a French cohort study, another SCN patient with the variant (p.Ala57Val) required high-dose G-CSF. After receiving a non-relative HSCT (4/6 matched), the patient remained well during a 10.3-year follow-up [25]. A Chinese girl with c.170 C > T (p.Ala57Val) missense variant has experienced recurrent upper respiratory tract infections since birth. With G-CSF and anti-infective treatment, her condition stabilized, and during follow-up, ANC fluctuated between (0.32–0.5)×10^9/L [26]. The Ala57 residue is a mutational hotspot, as evidenced by other pathogenic mutations affecting nearby amino acids (according to the ACMG classification criteria, including PM1). All previously reported missense mutations (Ala57Ser/Thr/Val) have been associated with SCN, except a CyN patient with Ala57Asp. However, our patient was diagnosed with an intermediate phenotype of CN. Thus, the present case highlights phenotypic variability in ELANE-related neutropenia due to an Ala57 mutation. Furthermore, our patient did not undergo G-CSF treatment. We have been conducting frequent monitoring of the ANC and hsCRP levels, and have been proactively using prophylactic antibiotics if the child presented with fever, oral ulcers, and hsCRP > 20 mg/L, or hsCRP > 40 mg/L even in the absence of fever or oral ulcer. Because CRP is a marker of the inflammatory process, and elevated CRP levels are significantly associated with bacterial infections, although the cutoff values for CRP may vary among different studies. However, there is currently a general consensus that a critical value of 20 mg/L for CRP limits the number of false negatives, ensuring appropriate treatment for patients with severe bacterial infections. A critical value of 40 mg/L for CRP reduces unnecessary antibiotic prescriptions [27]. During follow-up, there were no readmissions due to severe infections, and the patient’s condition remained favorable. As is known, the fundamental treatment for SCN involves G-CSF and anti-infective therapy, with HSCT being the optimal treatment for SCN patients with poor response to G-CSF [21, 28]. Moreover, a previous study has reported on p.Ala57Val variant leading to the manifestation of MDS/AML [2]. Our patient didn’t experience MDS/AML events, but it is necessary to pay attention to the risk of MDS or acute leukemia associated with the mutated Ala57 residue [29].

The neutropenia caused by ELANE variants poses a clinical challenge, and clinicians should be vigilant, particularly in diagnosing non-classical phenotypes, although the guideline in 2023 emphasized practical recommendations on diagnosis, classification, risk stratification, and prognosis, including the use of tests such as bone marrow morphology, flow cytometry, and cytogenetics [11]. Due to the challenges in diagnosing and treating this disease, we propose some hypotheses as follows: as is known, a proportion of primitive lymphoid cells > 20% in the bone marrow is required to diagnose acute lymphoblastic leukemia; similarly, it is necessary to collect more data on the proportion of promyelocytes and/or metamyelocyte in bone marrow cytology examinations of SCN and CyN patients to determine thresholds, aiding in diagnosis differentiation. Furthermore, we assume that it is feasible to utilize flow cytometry (FC) or fluorescence to measure NE activity to distinguish between SCN and CyN. International patient registries, such as SCNIR, and collaborative research networks globally to facilitate data sharing and utilizing indicators such as age at diagnosis, gender, cytogenetics, and molecular testing to construct risk prediction models are essential for prognosis stratification and targeted therapy, which will be an important future direction. When SCN patients experience fever, the appropriate use of antibiotics and G-CSF therapy is reasonable [21, 28]. The lack of response to G-CSF due to family rejection was a dilemma in clinical work and a limitation of our case. The patient had recurrent infections due to persistent neutropenia. It is still a clinical challenge to manage CN patients who refuse to use G-CSF and reduce the risk of severe infection. Long-term follow-up and frequent CBC monitoring are essentially crucial. Importantly, our case suggests that monitoring inflammatory markers can be used for guiding management. This management strategy was used by the clinical team in the absence of G-CSF use and is an area for future study. It may be an area for further research to determine whether monitoring inflammatory markers is useful in the prophylactic use of antibiotics, reducing the risk of hospitalization due to severe infections or improving prognosis. With limited time, the long-term prognosis of the child remains unclear.