Congenital neutropenia (CN) is a heterogeneous disorder. More than 30 distinct genetic defects have been discovered in patients with genetic diseases associated with decreased numbers of peripheral neutrophil granulocytes. Currently, most molecular diagnostic laboratories use Sanger-based sequencing techniques to define disease-causing mutations in patients with CN. In approximately 50% of patients no known genetic disorder can be found.

To identify novel genes that can be causative for unexplained CN cases we embarked on next-generation whole-exome sequencing using SOLiD 5500™ and Ion Proton™ sequencers. Up to date we sequenced whole exomes of 49 families, in which children were diagnosed with CN. The fragment libraries were constructed using the SureSelect™ V4+UTRs System (Agilent) allowing us to target whole coding sequence and the majority of UTRs of human genome (approx. 71 Mb). The vast majority of the families were analysed in the “Trio” approach and suitable homozygous or compound heterozygous rare variations (frequency below 1%) in protein coding regions or in splice sites were chosen for further validations.

In seven cases mutations previously described as causative for neutropenia were identified including G6PC3, HAX1, and ELANE. Four other rare variants are currently being analysed for their potential to cause CN. In 35 patients, no plausible candidate could be identified so far.

When we assessed variants within the genes related to CN, our data revealed unequal coverage pattern over these genes. Around 10% of the exons were insufficiently covered (coverage of less than 10) to allow for reliable variant and genotype call. These facts limit the power of whole exome sequencing as a diagnostic tool, as mutations at the non-covered positions cannot be ruled out, and demonstrate the need of an alternative comprehensive approach.

We are currently assessing sensitivity and specificity of a robust, rapid, and cost-effective approach that comprehensively analyses the sequence of 34 CN-relevant genes. Our approach is based on enrichment of specific exon regions by amplification using custom made AmpliSeq™ (Life Technologies) panel. For 25 genes we are able to sequence coding region as well as both UTR sequences, for 9 genes sequencing is limited to coding regions. This approach will provide a reliable, quick, and inexpensive diagnostic strategy for CN patients which will be offered free-of-charge to patients worldwide, independent of ethnic, national, or financial considerations.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.