ELAstase-Neutrophil Expressed (ELANE) gene is located on chromosome 19p13.3 and consists of five exons and four introns. Its DNA sequence consists of about 5000 base pairs. ELANE gene encodes neutrophil elastase (NE), a chymotrypsin serine esterase synthesized during the transformation of myeloblasts to promyelocytic granulocytes. NE is part of the NSP complex (NSPs), which also includes proteinase 3 (PR3) and histibin G [1–3]. NSPs only expressed in mature myeloid monocytes and play a key role in promoting inflammatory responses and the destruction of pathogens. Activation of the misfolded NE protein product and subsequent unfolded protein results in accelerated apoptosis during myeloid cell differentiation, followed by arrest of neutrophil precursor maturation at promyelocytic/granulocyte stage.
Intracellularly, NE and lysosomes participate in microbial decomposition synergized with antimicrobial peptide and NADPH oxidase system. Extracellularly, NE uses a variety of Gram-negative bacilli as substrates to split their pathogenic factors. In inflammatory reactions, NE destroys bacterial and host tissues. Therefore, NE also plays an important role in non-infectious inflammatory reactions. Chromatin binds to NSPs with positive charge or histones to form NETs. NETs capture pathogens through physical barriers, and NETS-related NSPs kill pathogenic microorganisms by degrading their virulence factors. Therefore, the lack of NE will lead to weakening of the organism's ability to clean pathogens.
The main clinical manifestation of ELANE gene mutation is neutropenia. It has been reported that mutations in the ELANE gene are found in 80 to 100% cases of cyclicneutropenia (CN) [4–6] and 35 to 63% cases of severe neutropenia (SCN) [6–8], both spontaneous and germline mutations occurred. ELANE gene mutation associated with neutropenia are usually frame shift and termination mutations that result in structural changes in the protein.
CN is an autosomal dominant disease characterized by regular fluctuation of peripheral neutrophils from near normal to severe low levels, generally with a cycle of 21 days, but also can be between 2–4 weeks [9]. Clinical manifestations include fever, lymphadenitis, oral ulcer and infection, including sinusitis, pharyngitis, cellulitis, pneumonia, acute peritonitis and so on [10–12]. The pathogen of infection is usually Gram-negative bacilli. Symptoms usually recur, but severe infections, sepsis, and even death is rare. It can also be asymptomatic. SCN is a congenital phagocytic defect in primary immunodeficiency diseases, which can be autosomal dominant or recessive inheritance. Spontaneous mutations have been reported more than germline mutations. It is characterized by repeated severe infection starting from first few months after birth. It can appear as omyelitis at first, followed by otitis media, pneumonia, skin or liver abscess. Patients can have permanent teeth fall at very early age due to stomatitis and gingival hyperplasia. Neutrophil absolute value (ANC) was always lower than 0.2×109/L[12, 13]. The most common pathogens are Staphylococcus aureus and Gram-negative bacilli. Because patients cannot produce pus, the absence of pus at infected lesions is one of its characters. Bone marrow biopsy shows neutrophil precursor maturation stop at the promyelocytic/myeloid stage. Unlike CN, SCN is characterized by transition to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) [1, 14].
In our study, case 1 presented with recurrent fever, infection and pediatric systemic lupus erythematosus. Her SLE was characterized by blood and kidney involvement, hypocomplement, and many autoantibodies positive, including high ANA, anti-SM, anti-SSA, anti-U1RNP, lupus anticoagulant and anti-β2 glycoprotein Ⅰ-IgG. Case 2 presented with repeated infection and autoimmune hemolytic anemia. Case 3 presented with recurrent infection and ANCA-associated small vasculitis. The main manifestations of his ANCA-associated small vasculitis were pulmonary involvement and positive P-ANCA. All three patients had recurrent fever and infection. Case 1 had recurrent episode, his fever and infection present at the same time, along with neutropenia. All three cases mainly had bacteria infection, and cases 1 and 3 also had Epstein-Barr virus infection. Different from previous reports, none of them had oral ulcer. According to literature study, ELANE gene mutation can associated with arthritis and pyoderma gangrenosum, but all of them were reported as individual cases. There were no reports of SLE, ANCA-associated small vasculitis and autoimmune hemolytic anemia. It has been reported that SCN caused by ELANE gene mutation can lead to MDS or AML. However, in our study, cases 2 and 3 were presented as SCN, but no MDS or AML on bone marrow biopsy. All 3 cases in this group had germline mutations, and case 1 also had NF1 gene mutation. The ELANE mutation of case 1 came from his father, who also presented with CN, but no recurrent infection. The parents of cases 2 and 3 had no clinical presentation of CN or SCN.
Recombinant human granulocyte colony-stimulating factor (G-CSF) can increase ANC level to normal or nearly normal in CN patients. Low-dose G-CSF treatment can shorten the lasting time of the lowest ANC value and increase the average ANC value, but it can not change the periodic change of ANC. G-CSF is the first treatment of choice for SCN. The Severe Chronic Neutropenia International Registry (SCNIR) reported that more than 95% of patients respond to G-CSF treatment and ANC can increase to 1×109/L. Most children with SCN respond to 3-10ug/kg.D [15–17]. For patients who do not respond to G-CSF, hematopoietic stem cell transplantation is the only treatment at present [18]. The incidence of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) is 40% after 10 years in SCN patients treated with G-CSF greater than 8ug/kg. However, this complication is relatively rare in patients with CN.
Although CN and SCN are mainly caused by ELANE gene mutations, it has been reported that different site of ELANE gene mutations can cause different clinical phenotypes, which means the presentation of CN or SCN depends on the site of ELANE mutation and its effect on NE activity. The genotype and phenotype may have one-to-one correlation. In this study, the three children had different genotypes, which led to different amino acid changes. Although two of them had SCN, they had different autoimmune diseases. The other case had CN, and the combined autoimmune disease was also different from the other two cases, which was consistent with the possible one-to-one relationship between genotype and phenotype reported previously.
By searching PUBMED, we did not find clear pathogenesis of ELANE gene mutation combined with autoimmune diseases. However, it has been shown that dysregulation of innate immune pathways related to host defense has profound effects on various aspects of SLE pathogenesis, including disruption of immune tolerance, induction of interferon and other proinflammatory cytokines, abnormal adaptive immunity, and tissue damage. Evidence found in human-mouse models, both in vivo and in vitro, supports that neutrophil dysregulation plays a key role in the pathogenesis of SLE, including loss of immune tolerance, induction and amplification of inflammatory pathways, tissue damage, vascular disease, and cardio metabolic dysfunction [19–22]. It has been reported that genetic diseases which change the cellular components of innate immunity have been shown to increase the risk and severity of SLE. ELANE gene mutation leads to neutropenia, which plays an important role in innate immunity, leading to reduced anti-bacterial ability and weakened effect in coagulation, angiogenesis, inflammation resolution and tissue repair. In addition, neutrophils can regulate innate and adaptive immune cells. The three children in this study were combined with SLE, AIH and ANCA-related small vasculitis, respectively. These three diseases are all systemic damage caused by autoantibodies. Therefore, AIH and ANCA-related small vasculitis may also have a similar mechanism as SLE.
Case 1
showed CN phenotype. Oral Rikedron could alleviate the degree of neutropenia and subsequently improve the susceptibility to infection. At the same time, methylprednisolone, hydroxychloroquine and cyclosporine were used for SLE. The patient was followed up for more than 2 years. His fever and recurrent infection reduced, and the dose of methylprednisolone was reduced successfully. Patient 2 had no recurrent infection after allogeneic hematopoietic stem cell transplantation, and was followed up for more than 2 years. Her Coombs test turned negative and neutrophils was normal. In case 3, subcutaneous injection of G-CSF improved neutropenia, and methylprednisolone and cyclophosphamide were given for ANCA-associated small vasculitis. After follow-up for more than one year, he did not have severe infection. His p-ANCA turned negative, and pulmonary lesions gradually disappeared. ELANE gene mutation combined with autoimmune diseases is rare. In the treatment of neutropenia, appropriate prednisolone and immunosuppressive therapy can be selected according to different autoimmune diseases. In addition, allogeneic hematopoietic stem cell transplantation can fundamentally change the immune deficiency and immune disorder in patients, and achieve satisfactory effect in children with poor response to G-CSF or combined with MDS/AML.