Role of genetic testing in diagnosing patients with severe congenital neutropenia: Results from PATH4WARD genetic testing program

Abstract Patients with suspected severe congenital neutropenia (SCN) frequently exhibit a wide spectrum of clinical manifestations, which can complicate timely and accurate diagnosis. Following the exclusion of acquired etiologies, genetic testing should be incorporated into the diagnostic evaluation for individuals with a clinical suspicion of congenital neutropenia. In this report, we present findings from PATH4WARD, an X4 Pharmaceuticals-sponsored genetic testing initiative aimed at supporting early and precise molecular diagnosis of primary immunodeficiencies (PIDs) associated with neutropenia. Identification of these conditions may facilitate appropriate clinical management, initiation of approved therapeutic interventions, and consideration for enrollment in appropriate interventional clinical trials. The PATH4WARD program, conducted between July 2019 and April 2025, utilized a next-generation sequencing (NGS) approach with panel expansion over time. Initially launched with a 23-gene SCN panel in 2019, the testing was expanded to a 407-gene primary immunodeficiency (PID) panel in 2021, and further extended in 2022 to a comprehensive 574-gene panel encompassing both PID and cytopenia-associated genes. Variant interpretation was performed using Sherloc, an evidence-based classification system based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines. Of 3,088 individuals who underwent testing, 355 (11.5%) received a positive molecular test result. 93 (3%) patients were diagnosed with SCN with pathogenic or likely pathogenic variants in 11 unique genes (ELANE, CXCR4, G6PC3, TCIRG1, GATA2, HAX1, TAZ, WAS, CSF3R, VPS13B, and CD40LG). The most common type of SCN-causing variants identified were ELANE (48 patients), followed by CXCR4 (18 patients), G6PC3, and TCIRG1 (5 patients each). 267 (8.6%) patients received a positive molecular test result for another type of PID (5 of these patients received positive test results for both SCN and another PID). To illustrate the clinical utility of genetic testing in guiding diagnosis and treatment decision making, we present the case of a patient with neutropenia who underwent genetic evaluation through the PATH4WARD program. In 2022, at age 11 years, the male patient was found to harbor CXCR2 c.865C>T, p.Arg289Cys, a variant associated with SCN due to CXCR2 deficiency.1 In 2023 the patient was enrolled in a Phase 1b and Phase 2 clinical trial investigating mavorixafor in participants with congenital neutropenia and chronic neutropenia disorders, including chronic idiopathic neutropenia (NCT04154488). The participant was receiving G-CSF background therapy for chronic neutropenia, and during the study, the investigator and participant chose to reduce the G-CSF dosage, achieving a 50% reduction by month 4, which was maintained through month 6. Despite this substantial reduction in G-CSF, the patient's absolute neutrophil count remained at or above approximately 1000 cells/µL throughout the study. This case highlights the value of genetic testing in establishing a molecular diagnosis and enabling access to investigational agents through clinical trial participation, with the potential to improve clinical outcomes. Reference:Marin-Esteban, et al. Haematologica. Mar 1 2022;107(3):765-769.