Extended Genetic Testing in Severe Congenital Neutropenia May Identify Mutations That Inform Therapy

The Severe Chronic Neutropenia International Registry (SCNIR) in cooperation with Washington University has developed a next-generation sequencing assay for the genetic diagnosis of chronic neutropenia. Exome-based sequencing is performed, and genetic alterations in 27 genes implicated in the pathogenesis of severe congenital neutropenia (SCN) are reported. Of note, with consent, patients are enrolled in the SCNIR to collect detailed clinical data and to allow researchers access to the exome sequence data to identify new genetic alterations contributing to disease pathogenesis, severity, and progression to acute myeloid leukemia. Here, we report sequencing results for 61 cases of SCN, 31 cases of cyclic neutropenia, and 3 cases of idiopathic or unclassified neutropenia. The median age of this cohort was 17.2 years (range 0.13 to 78 years). Ninety-eight per cent of the SCN cases received G-CSF with a median dose of 1.85 mg/kg/day (range, 0-99.3), and 87% of cyclic neutropenia cases received G-CSF with a median dose of 0.34 mg/kg/day (range, 0-5.2). ELANE mutation status was previously determined for the majority of these cases. In total, 16 of 95 cases of neutropenia had a known ELANE mutation, and the remainder of the cases had no genetic diagnosis.

Initial analyses focused on the following genes previously implicated in SCN pathogenesis: ELANE, AP3B1, CSF3R, CXCR2, CXCR4, G6PC3, GFI1, HAX1, LYST, LAMTOR2, RAB27A, SBDS, SLC37A4, SRP54, TCIRG1, VPS13B, VPS45, USB1, TAZ, and WAS. Heterozygous mutations of ELANE were identified in the 16 previously identified cases, but in no additional cases. Of the remaining 50 cases of SCN, probable pathogenic mutations were identified in 8 (16%). Surprisingly, in the 27 cases of cyclic neutropenia with wildtype ELANE, no probable pathogenic mutations were identified. We identified three cases of SCN (including two family members) with biallelic mutations of G6PC3 and single cases of SCN with N382S GFI or R184C TCIRG1 mutations. A novel homozygous R547X CSF3R mutation was seen in one case of SCN. This truncation mutation in the extracellular domain of the G-CSF receptor is predicted to result in a complete loss-of-function allele. Indeed, this patient was refractory to G-CSF therapy. We also identified a single case of SCN carrying a R338X CXCR4 mutation, the cause of WHIM syndrome. Of note, emerging data suggest that the neutropenia in WHIM syndrome can be effectively treated with CXCR4 antagonists. We identified two cases of SCN carrying heterozygous mutations of SRP54, G175E and T191I, respectively. These SRP54 mutations, although not previously reported, localize to the GTPase domain of SRP54 (similar to previously identified SRP54 mutations), and they have not been identified in over 60,000 individuals in the ExAC database.

Conclusions: Extended genetic testing in patients with chronic neutropenia and wildtype ELANE, although identifying a pathogenic mutation in the minority of cases, may impact the choice of therapy. Analyses of the exome sequence data are underway to identify novel candidate genes contributing to neutropenia.