P791: whole exome sequencing in adult patients with chronic idiopathic neutropenia

Topic: 12. Bone marrow failure syndromes incl. PNH - Clinical Background: Adult patients with persistent, unexplained neutropenia who do not fulfill the diagnostic criteria of any underlying disease are characterized as chronic idiopathic neutropenia (CIN) cases. It is a diagnosis of exclusion that can be established after a thorough clinical/laboratory investigation for any underlying causes, including negative anti-neutrophil antibody testing, inconclusive bone marrow (BM) aspiration/biopsy and normal cytogenetics. CIN patients regardless of their absolute neutrophil counts (ANC) (most often presented with mild/moderate and rarely with severe neutropenia) usually display a benign and uncomplicated clinical course. Although the pathogenesis of CIN remains largely unknown, we hypothesize that a constitutional/congenital background might exist, at least in some cases, that have potentially escaped diagnosis during childhood. Aims: The aim of the study is to investigate for a potential genetic background of neutropenia in CIN by conducting whole exome sequencing (WES) analysis in a number of adult CIN patients. Methods: We have performed WES in 16 adult (median age 59 years, range 30-72 years) patients (11 females and 5 males) with CIN according to previously published criteria, i.e. no evidence of any underlying condition related to neutropenia after an extended clinical/laboratory investigation including BM biopsy, karyotype, immunophenotype. The patients had a median ANC 1.2 x 109/L, range 0.4 -1.6 x 109/L, for prolonged period and unknown neutropenia onset in most cases. Genomic DNA was extracted from peripheral blood samples, coding fragment libraries were prepared, sequencing performed on Illumina NovaSeq 6000 System, and data subjected to an in-house pipeline for bioinformatic analysis for variants calling and annotations. Afterwards, multi-samples and singleton analyzes were performed. Mean depth of coverage was 70x and the mean number of variants/sample was 22553. Results: WES analysis identified nine pathogenic/likely pathogenic variants in six patients (Table 1). No known pathogenic variants in genes associated with Congenital Neutropenia were detected in our cohort apart from one patient bearing a single heterozygous mutation in G6PC3 gene (previously detected in one of our patients with Congenital Neutropenia; Nikolouzakis TK et al, Ann Hematol. 2022). Along with pathogenic and likely pathogenic gene variants, several variants of unknown significance (VUS) were also detected, consisting of probably deleterious variants in genes associated with BM failure (GATA2, SAMD9, CXCR4), severe combined immunodeficiency (ORAI1), neutrophil dysfunction (RASAL3) or agammaglobulinemia (TCF3) (Table 1). However, none of our patients display clinical symptoms typical of these conditions and since the majority of VUS are detected in genes not yet known to be associated with the neutropenia phenotype, they will be further investigated for their potential contribution to the disease onset. Summary/Conclusion: To our knowledge, this study is the first attempt of WES analysis in adult CIN patients. The identified P/LP variants and VUS will be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to the neutropenia phenotype. Overall, this study contributes to the better understanding of the pathogenesis of this puzzling disease.Keywords: Neutropenia, Mutation analysis