Analysis of Genetic Variants in Myeloproliferative Neoplasms Using a 22-Gene Next-Generation Sequencing Panel

Background: The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) (i.e. essential thrombocythaemia (ET), polycythaemia vera (PV), and primary myelofibrosis (PMF)) are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these Ph-negative MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of Ph-negative MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 44 variants with minor allele frequencies (MAFs) of ≥ 1%, and 20 variants with MAFs of < 1% were identified (4 of which were putatively novel variants with potential biological significance) across the 10 MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants support its potential clinical utility in the management of Ph-negative MPNs.