Screening of genetic variants in mutation negative congenital neutropenia by next generation sequencing.

Aims: Congenital neutropenia (CN) is a rare inherited disease that results in recurrent, life-threatening bacterial infections due to a deficiency of mature neutrophils. They are usually caused by heterozygous mutations although mutations in other genes like and have also been reported. Identifying the causative mutation aids in the establishment of diagnosis and rules out other secondary causes of neutropenia like autoimmune cytopenia and evolving aplasia. We aimed to identify the molecular defects in CN patients who had no mutations in gene, by next generation sequencing (NGS) targeting a customised panel of genes.

Methods: DNA samples were sequenced with an Illumina NextSeq sequencer using an in-house customised panel of genes at ≥100× depth. Bioinformatics analysis was carried out and the pathogenic variants were identified using a stepwise filtering and analysis strategy. Specific mutations identified were subsequently validated by Sanger sequencing.

Results: The pathogenic variants identified in the study includes previously reported variants in (compound heterozygous c.258+2T>C and c.1A>T), (heterozygous c.1186C>T) and novel variants in (hemizygous c.812T>C), (homozygous c.70G>A) and (heterozygous c.2893G>C) genes.

Conclusion: This study highlights that the absence of mutations does not rule out the diagnosis of CN and this NGS based approach with a customised panel will help in diagnostic confirmation in such patients. The early onset of the disease, clinical severity and associated high risk of malignant transformation in CN strongly suggests the need for early diagnosis and therapeutic intervention.