Absence of Neutropenia in Patient with an Exon 2 Nonsense Mutation in ELANE: Clinical Evidence to Support Gene Therapy Efforts for Severe Congenital Neutropenia
Authors: Joos Margret , Shimamura Akiko , Chang Timothy H. , Newburger Peter E. 
Published: December 2023

Introduction
Severe congenital neutropenia (SCN) is an inherited bone marrow failure condition most often caused by heterozygous mutations in the ELANE gene. While granulocyte colony stimulating factor (G-CSF) is effective in increasing neutrophil counts in the majority of patients, approximately 15% of patients do not respond, necessitating the creation of alternative treatment strategies. Due to its association with significant, life-threatening infections and heightened risk of progression to myelodysplastic syndrome and/or acute myeloid leukemia, SCN has been the focus of recent gene therapy efforts by multiple groups (Nasri et al., 2020; Rao et al., 2021).
Pathogenic variants in ELANE most often cause missense mutations or in-frame indels which lead to the production and expression of mutant neutrophil elastase (NE) proteins. Frameshift mutations resulting in out-of-frame stop codons or nonsense mutations are also seen in the third through fifth (final) exons of ELANE due to their ability to circumvent nonsense-mediated decay (NMD) and generate mutant NE. In contrast, frameshift and nonsense mutations in the first and second exons have not been shown to cause neutropenia, most likely because NMD prevents the translation of mutant NE (Rao et al., 2021; Horwitz, 2021). Collectively, these observations suggest that the pathogenicity of ELANE mutations may rely on gain-of-function alterations in mutant NE rather than loss-of-function alterations in normal NE.
Because the absence of one copy of ELANE does not appear to be deleterious, one theory posits that therapeutic inactivation of mutant copies of ELANE may have corrective potential (Horwitz et al., 2019; Nasri et al., 2020; Rao et al., 2021). Thus, the focus of several gene editing efforts has been CRISPR/Cas9-mediated knockout in early exons of ELANE in hematopoietic stem and progenitor cells (HSPCs) of patients with ELANE-mutated SCN for allogeneic stem cell transplant (Nasri et al., 2020; Rao et al., 2021). With this study, we sought to understand whether an inherited ELANE mutation analogous to those utilized in gene therapy would cause neutropenia.
Case Presentation
Here, we present a six-month-old Caucasian male with an ELANE mutation whose clinical findings support this model. The patient was born at full term to a 35-year-old G6P2 mother. The patient's constellation of complex symptoms, including polymicrogyria, corpus callosum dysgenesis, hypotonia, seizures, and feeding difficulties prompted clinical trio whole genome sequencing (WGS) at approximately two months of age. The patient was incidentally found to have a de novo nonsense variant in exon 2 of the ELANE gene (NM_001972.2:c.151G&gt;T, p.[G51*]), predicted to result in protein truncation or NMD. WGS also identified variants of uncertain significance in CHRNB2 and HFE.
Importantly, although the ELANE variant was classified as likely pathogenic in the clinical genetics report, the patient has not had clinically significant neutropenia. Absolute neutrophil counts have ranged from 1.27 - 10.95 K cells/μL, with mean and median values of 4.09 and 2.95 K cells/μL, respectively (n=10). The patient has not had a significant infectious history, nor has he required any treatment for hematologic concerns.
Discussion
The absence of a neutropenia pathology in this patient, at least in his first six months of life, provides evidence in support of gene therapy efforts which seek to ameliorate pathogenic ELANE mutations with gene editing to produce frameshifts in early exons. Most likely, in this patient, NMD of mutant ELANE mRNA prevented its translation, thus allowing for ample production and maturation of neutrophils. A similar pattern was observed in another report which examined patients with whole gene deletions of ELANE (Horwitz et al., 2019), but this is the first report of a nonsense variant analogous to those proposed to be introduced by gene or base editing for gene therapy of SCN. Thus, the present case adds to a growing body of evidence which suggests that ELANE knockout may have therapeutic potential for SCN.