ASSESSMENT OF CONGENITAL NEUTROPENIA IN CHILDREN: COMMON CLINICAL SCENERIES AND CLUES FOR MANAGEMENT

A disparate group of rare hematological diseases characterized by impaired maturation of neutrophil granulocytes defines congenital neutropenias. Neutropenic patients are prone to recurrent infections beginning in the first months of life. Of interest is “cyclic neutropenia”, an ultra-rare disorder revealed by sinusoidal variations of the neutrophil count and periodically-recurring infections every 21 days. Diagnosis of these disorders is frequently obscured by the multiple causes of recurrent fevers in children. Aim of this overview is to outline the physical assessment of children presenting with early-onset symptomatic neutropenia, identify the disease between the many medical conditions and even emergencies which should enter in differential diagnosis, hint at the potential management with granulocyte-colony stimulating factor, define the risk of evolution to hematologic malignancy, and summarize inter-professional team strategies for improving care coordination and outcomes of such patients.

USB1 Is a miRNA Deadenylase That Regulates Hematopoietic Development

Poikiloderma with neutropenia (PN)is an autosomal-recessive bone marrow failure (BMF) syndrome in which patients harbor homozygous or compound heterozygous mutations in the human gene C16orf57, which encodes the evolutionarily conserved RNA 3' to 5' exonuclease U6 biogenesis 1 (USB1). USB1 is required for the proper maturation of U6 and U6atac snRNAs, core components of the spliceosome, and consequently, splicing defects have been observed in yeast and zebrafish models with USB1 deficiency. However, lymphoblastoid cells from PN patients do not exhibit reduced U6 snRNA levels and have normal pre-mRNA splicing, establishing PN as a singular BMF syndrome, where the underlying genetic cause has been identified but the molecular mechanisms leading to tissue failure remain obscure. To investigate the role of USB1 in a physiological context, we utilized CRISPR/Cas9 to create human embryonic stem cells (hESCs) containing a frequently occurring c.531_del_A loss-of-function mutation in the USB1 gene (USB1_c.531_del_A hESCs). USB1_c.531_del_A hESCs have normal karyotype, normal growth rate, and retain pluripotency, indicating that clinically-relevant mutations in USB1 are not deleterious in undifferentiated hESCs. To elucidate the role of USB1 during hematopoiesis, we performed serum-free hematopoietic differentiations to derive hematopoietic progenitor cells from WT and USB1_c.531_del_A hESCs. The formation of definitive hematopoietic progenitors (CD45+) was decreased in USB1 mutant cells compared to WT cells, and definitive colony potential analysis showed compromised colony formation in USB1 mutants. These observations indicate that loss-of-function mutations in USB1 negatively influence hematopoiesis. Additionally, as PN is associated with severe non-cyclic neutropenia, we analyzed the potential of neutrophil formation in WT and USB1 mutant cells. USB1 mutants have reduced formation of CD15+/CD66b+ lineages, indicating abnormal neutrophil development. Conditional expression of WT USB1 in USB1_c.531_del_A mutant cells rescued these phenotypes, leading to normal hematopoietic development. Interestingly, USB1 mutants showed no reduction in the overall levels of U6 and U6atac snRNAs, similar to what was observed in patient cells. To identify other possible targets of USB1, we sequenced the transcriptome and miRome of WT and USB1 mutant cells in different stages of hematopoietic development. Through these analyses, we demonstrate that hematopoietic failure in USB1 mutants is caused by dysregulated miRNA levels during blood development, due to a failure to remove destabilizing 3' end oligo(A) tails added by PAPD5/7. Moreover, we demonstrate that modulation of oligoadenylation through genetic or chemical inhibition of PAPD5/7 rescues the defective hematopoiesis observed in USB1 mutants. This work indicates USB1 acts as a miRNA deadenylase and suggests PAPD5/7 inhibition as a potential therapy for PN. Disclosures Parker: Faze Therapeutics: Other: Co-founder.

Spectrum of Pathogenic Genetic Variants in a Large Cohort of North American Congenital and Cyclic Neutropenia Patients: A Report from the Severe Chronic Neutropenia International Registry

Severe congenital neutropenia (SCN) is characterized by persistent neutropenia and risk of invasive, life-threatening infection as well as transformation to hematopoietic malignancy. The closely related syndrome cyclic neutropenia is characterized by recurrent episodic neutropenia accompanied by symptoms including infection. Understanding the genetic etiology of congenital neutropenia can help to direct therapy, guide surveillance and health maintenance strategies, and contribute to our understanding of basic neutrophil biology. Although mutations in ELANE are the most frequent cause of congenital neutropenia, there is a wide and ever-growing list of additional causative variants. Additionally, there appears to be regional genetic variability. For example, mutations in HAX1 are rarely if ever observed in North America while they are more common in Europe. We undertook exome sequencing of a large cohort from the Severe Chronic Neutropenia International Registry (SCNIR) of North America in an effort to define the genetic spectrum of congenital neutropenia and aid in the discovery of new pathogenic variants. We expanded our previously reported study of whole exome sequencing to include 152 cases of chronic neutropenia, comprised of 94 cases of SCN and 58 cases of cyclic neutropenia. We selected cases in which ELANE testing was negative, or in a small minority of cases, where ELANE testing had not yet been performed. Indeed, exome sequencing only identified 7 cases (5 SCN and 2 cyclic) carrying pathogenic ELANE mutations in this cohort. In the remaining 145 cases, we analyzed exomes for the presence of variants in genes previously associated with congenital neutropenia including AK2, AP3B1, CD40LG, CLPB, CSF3R, CXCR2, CXCR4, DNAJC21, DNM2, DOCK2, EFL1, EIF2AK3, ELANE, G6PC3, GATA1, GATA2, GFI1, GINS1, HAX1, IRAK4, JAGN1, KAT6A, KRAS, LAMTOR2, LYST, MYD88, PGM3, PSTPIP1, RAB27A, RAC2, SBDS, SEC61A1, SLC37A4, SMARCD2, SRP54, STK4, TAZ, TCIRG1, TCN2, TLR8, USB1, VPS13B, VPS45, WAS, WDR1 and WIPF1. Pathogenic heterozygous mutations of CLPB that localize to the ATP-binding pocket were identified in 7 cases, making it the second most common cause of congenital neutropenia in North America. We additionally identified 4 cases with G6PC3 pathogenic variants, and one case each with pathogenic variants in JAGN1, CXCR4 (the cause of WHIM syndrome), germline homozygous CSF3R, and GFI1. Interestingly, we identify 2 unrelated individuals (one with SCN and one with cyclic) and 2 siblings with SCN all of whom possess a recently described heterozygous variant in SRP54 (p.T117del). We collected genomic DNA from the affected mother of the 2 siblings, an additional unaffected sibling, and the unaffected grandparents. Through this kinship, we can confirm the de novo appearance of this variant in the second generation and demonstrate that it tracks with disease status (Figure 1). We also identified 2 unrelated individuals with SRP54 variants affecting residue 175 also located within the GTPase domain (p.G175E or p.G175del). Both variants are absent from the gnomAD database, and studies are underway to demonstrate de novo acquisition. In summary, we have defined the spectrum of mutations present in ELANE-wildtype chronic neutropenia cases in North America. Pathogenic or likely pathogenic variants were identified in 26 out of 145 (18%) cases. The most frequently mutated genes were of CLPB, SRP54, and G6PC3, while mutations in HAX1 were not seen. Importantly, some of these mutations are associated with genetic syndromes with extra-hematopoietic findings (for example, CLPB and SRP54) that would warrant additional evaluations and targeted health maintenance. These findings emphasize the importance of sending large panels for genotyping, rather than targeted ELANE testing. Figure 1 Figure 1. Disclosures Bolyard: X4 Pharmaceuticals: Research Funding. Makaryan: Emendo Biotherapeutic: Research Funding. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

ELANE Neutropenia Beyond the Classification into Cyclic Neutropenia and Severe Congenital Neutropenia: The Inconstant Time Clock

Introduction: ELANE neutropenia represents the cause of 25-30% of the cases of congenital neutropenia. Classically, its appears in the literature as in OMIM, under two distinct entities: Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN). The delineation between the 2 entities is the "cyclicity" i.e. the periodic variation of the absolute neutrophils count (ANC), also called a 21-day time clock 1. However, it is extremely difficult to obtain enough sequential complete blood counts (CBC) at the onset of the disease, during an enough length period, while the patient is not experiencing a severe infection or receiving GCSF therapy. The purpose of our study is to analyze the ANC periodicity at the onset of the disease, prior to the initiation of GCSF in a cohort of patients with ELANE neutropenia. Methods : Available data from the patients, with ELANE class 4 and 5 variants, enrolled in the French Severe Chronic Neutropenia Registry, were analysed. The final diagnostic of CyN and SCN was performed considering all the follow up period (median 16.7 years) and based on the presence of recurrent periodic variation of ANC in the absence of GCSF therapy. CyN was defined as multiple documented ANCs >500 cells/mm 3 , with intermittent ANC variation (n=49), while SCN is defined as ANC persistently <500 cells/mm 3 (n=94). In case of irregularity (i.e. not a regular periodic pattern during all the follow up), the classification takes in consideration the majority of the follow up. A comprehensive analysis of the infectious profile is available elsewhere 2. We were focused here on the diagnostic period (roughly the 2 first months since the diagnosis). We have analysed the initial blood count of the patients and cast the patients by categories if at least 4 ANCs can be evaluated. ANC oscillations defined 4 groups: Group 1: oscillation of ANC values above and below 500 ANC/mm 3 for at least 2 cycles lead to consider the patient as Cyclic; Group 2: clear oscillation of ANC values above and below 200 ANC/mm 3 (but ever<500 ANC/mm 3) for at least 2 cycles; Group 3: no oscillation of ANC values whose level are ever below 500 ANC/mm 3; Group 4: Early GCSF treatment. Results : Among the 143 patients enrolled in this study (Table 1), 137 have at least 4 CBC evaluable during the diagnosis period, including 30 who have been treated almost front line after diagnosis of neutropenia by GCSF hampering evaluation of periodicity. Such patients were all initially considered as SCN. Among the 67 finally classified as SCN, 28 (27.38%) showed an oscillation pattern (group 1), 14 (15.48%) showed minor oscillations (group 2), while 25 (30.95%) had a persistent and severe neutropenia (group 3). Among the 40 CyN, 1 have showed minor oscillations (group 2) below 500/mm 3, while 3 had a persistent and severe neutropenia (group 3). Globally, 32 /107 patients were miss- classified at diagnosis compare to the final diagnosis. Additional data shows that many health indicators could not be deducted from the initial classification like the infections rate, the use of GCSF, the death rate, the sequels rate. Conclusions: Periodic variation of ANC despite being the criteria to define the sub type of ELANE neutropenia is difficult to evaluate at the initial presentation of the disease. In addition, cyclicity is not a permanent feature in ELANE neutropenia, some patients being cyclic only for a certain time in their life span. It results a high rate of miss classification if we compare the initial diagnostic period and all the medical history of the patients. Noteworthy, the rate of several severe complications is not so clearly different between diagnosis sub categories. We propose to consider ELANE neutropenia as a unique disease characterized by a clinical spectrum ranging from more severe forms (corresponding to SCN) to milder forms, the latter often characterized at onset by ANC fluctuations. In addition, some intermediate severity forms could be characterized by minor oscillations. References 1 Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat.Genet. 1999;23:433-436. 2 Rotulo GA, Plat G, Beaupain B et al. Recurrent bacterial infections, but not fungal infections, characterise patients with ELANE-related neutropenia: a French Severe Chronic Neutropenia Registry study. Br J Haematol. 2021 doi: 10.1111/bjh.17695 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A Novel Pathogenic Variant of G6PC3 Gene Presenting As Cyclic Neutropenia in a Pediatric Patient

Background: Cyclic neutropenia is a rare hematologic disorder affecting neutrophils. It is classified by recurrent neutropenia (typically every three weeks) with associated symptoms including fever, malaise, and recurrent mucosal and skin infections. Pathogenic variants of ELANE, the gene that encodes neutrophil elastase, are thought to cause these findings, however, other pathogenic variants of different genes have also been noted. The estimated frequency of cyclic neutropenia is 1/10 6 in the general population. It is most commonly diagnosed in children but may rarely be diagnosed in adulthood. There is no known increased prevalence in women as compared to men. Whole Exome Sequencing can be used for diagnosis when genetic concerns are present. Objective: This case report aims to describe a novel pathogenic variant in a pediatric patient with cyclic neutropenia. Design/Method: Single subject case report Results: This is a single case report of a 7-year-old female with past medical history of cyclic neutropenia, anemia, recurrent skin and buccal infections, speech delay, and behavioral problems. The patient initially presented at age 1 with high fevers and skin infections and was found to have decreased neutrophil counts. Work up at that time was consistent with cyclic neutropenia. Bone marrow aspirate and biopsy performed at 1 and 7 years of age showed decreased neutrophil count with normal maturation without any evidence of MDS. Her peripheral blood for bone marrow failure evaluation was non-contributory. Due to persistent neutropenia, normal bone marrow findings, and being on weekly chronic G-CSF administration Whole Exome Sequencing was evaluated. Results of which showed a novel compound heterozygous pattern for two variant copies of the G6PC3 gene, with our patient inheriting a copy from each parent. Discussion: The G6PC3 gene encodes the expressed glucose-6-phosphate enzyme which catalyzes the final step in glycogenolysis. It is hypothesized that this deficiency causes unregulated levels of glucose, resulting in increased stress of the endoplasmic reticulum leading to apoptosis of neutrophils. Pathogenic variants of G6PC3 cause autosomal recessive G6PC3 deficiency and this can be clinically characterized as severe congenital neutropenia. Classic G6PC3 deficiency includes severe congenital neutropenia as well as cardiovascular abnormalities, urogenital abnormalities, and pulmonary hypertension. This novel pathogenic variant is likely responsible for the cyclic neutropenia observed in our patient. G6PC3 variants are an important differential diagnosis in the work-up of other causes of neutropenia. Whole Exome Sequencing is a cost-effective method for diagnosis and a valuable tool in evaluation and management of complex hematologic disorders. Disclosures No relevant conflicts of interest to declare.

Elamipretide for Barth syndrome cardiomyopathy: gradual rebuilding of a failed power grid

Barth syndrome is a rare and potentially fatal X-linked disease characterized by cardiomyopathy, skeletal muscle weakness, growth delays, and cyclic neutropenia. Patients with Barth syndrome are prone to high risk of mortality in infancy and the development of cardiomyopathy with severe weakening of the immune system. Elamipretide is a water-soluble, aromatic-cationic, mitochondria-targeting tetrapeptide that readily penetrates and transiently localizes to the inner mitochondrial membrane. Therapy with elamipretide facilitates cell health by improving energy production and inhibiting excessive formation of reactive oxygen species, thus alleviating oxidative stress. Elamipretide crosses the outer membrane of the mitochondrion and becomes associated with cardiolipin, a constituent phospholipid of the inner membrane. Elamipretide improves mitochondrial bioenergetics and morphology rapidly in induced pluripotent stem cells from patients with Barth syndrome and other genetically related diseases characterized by pediatric cardiomyopathy. Data with elamipretide across multiple models of disease are especially promising, with results from several studies supporting the use of elamipretide as potential therapy for patients with Barth syndrome, particularly where there is a confirmed diagnosis of cardiomyopathy. This review highlights the challenges and opportunities presented in treating Barth syndrome cardiomyopathy patients with elamipretide and addresses evidence supporting the durability of effect of elamipretide as a therapeutic agent for Barth syndrome, especially its likely durable effects on progression of cardiomyopathy following the cessation of drug treatment and the capability of elamipretide to structurally reverse remodel the failing left ventricle at the global, cellular, and molecular level in a gradual manner through specific targeting of the mitochondrial inner membrane.

ELANE neutropenia and solid tumors: 4 cases from the French severe chronic neutropenia registry

Neutropenia related to ELANE gene mutations predisposes to infection and leukemia/ myelodysplasia, but little is known about the predisposition to cancer. Among a cohort of 149 patients, we identified four with malignant solid tumors (papillary thyroid cancer, anal squamous cell cancer, papillary renal cell carcinoma, and adrenocortical carcinoma), all after the age of 25 years. Three occurred in cyclic neutropenia while 1 occurred in severe chronic neutropenia (among 49 and 100 patients, respectively). A previous radiotherapy was identified as risk factor in one patient. Moreover, among 18 other patients that underwent hematopoietic stem-cell transplantations, none developed a cancer.