Assessment of the Frequency of Variants of Undetermined Significance (VUS) in Adult Patients with Chronic Idiopathic Neutropenia Studied for Myeloid Gene Mutations By Next Generation Sequencing

Background: Chronic Idiopathic Neutropenia (CIN) is a neutrophil disorder characterized by the persistent and unexplained reduction in the number of peripheral blood (PB) neutrophils. The diagnosis of CIN is based on exclusion criteria, namely the absence of clinical/laboratory evidence of any underlying disease or use of drugs that might be associated with neutropenia, absence of history of exposure to irradiation or use of chemical compounds, negative antineutrophil antibodies, and normal bone marrow (BM) morphology and karyotype. We have previously performed next generation sequencing (NGS) analysis of genes recurrently mutated in myeloid malignancies in a cohort of CIN patients and we found that 11.35% of the patients display clonal hematopoiesis in term of presence of pathogenic/likely pathogenic variants with VAF higher than 2%. Variants of undetermined significance (VUS) were ruled out from the analysis and their potential significance was not assessed.

Aims: In the current study we have assessed the presence, frequency and potential clinical significance of VUS detected in our cohort of CIN patients previously studied for clonal hematopoiesis.

Methods: Genomic DNA was extracted from CIN patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted NGS on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies (Tsaknakis G et al. Blood. 2021;138:1249-1257).

Results: NGS analysis of myeloid genes has been performed in 139 CIN patients (106 female subjects and 33 male subjects, aged 21 to 89 years with a median age of 59 years). Re-analysis of patients' NGS data and assessment of VUS revealed that 35 out of 139 patients (25.18%) carried one or more VUS (a total of 39 VUS were detected, all with an allelic frequency of 50% and all missense variants). Genetic variants were evaluated according to the ACMG guidelines and classified as VUS. The majority of VUSs were observed in ATM (n=7) and SETBP1 (n=6) genes followed by TET2, NOTCH1 (4 occurrences respectively) and PLCG2 (n=3). Two different patients shared the same variant in TET2 gene whereas two siblings shared the same variant in SETBP1 gene. Pathogenicity score calculation based on the combined evidence from multiple other in-silico predictors identified 3 variants in 3 different genes as having the highest probability to be pathogenic. Namely these were PLCG2^K456E,CXCR4^R235C and SF3B1^T56I. PLCG2 variants have been implicated in familial cold autoinflammatory syndrome 3 (PLCG2 associated antibody deficiency and immune dysregulation, PLAID) and in autoinflammation, antibody deficiency, and immune dysregulation syndrome (APLAID) whereas CXCR4 variants are known to be associated with Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. SF3B1 mutations have not yet been shown to be involved in hereditary disease.

Conclusions: Our study of VUS assessment in a cohort of 139 well characterized adult CIN patients identified 39 VUS, all heterozygous, in a total of 35 patients. Further steps include paired analysis with constitutional reference tissue to determine the germline nature of the mutations. Moreover, these variants should be functionally tested and integrated with functional results and clinical phenotypes to more accurately interpret their potential contribution to disease onset. This study of VUS analysis in CIN patients is anticipated to contribute to the better understanding of the pathogenetic causes of this rare disease.

No relevant conflicts of interest to declare.