Resolving diagnostic uncertainty in rare MPL and CALR variants: A practical framework for the haematology clinic

Interpretation of rare non-canonical MPL and CALR variants in myeloproliferative neoplasms (MPNs) remains challenging and frequently results in variants of uncertain significance (VUS). We explored whether these variants share structural or electrostatic features with established MPN driver mutations. We studied 179 patients with BCR::ABL1-negative MPNs using targeted next-generation sequencing (NGS), structural modelling, in silico pathogenicity assessment and unsupervised phenotypic clustering. MPL variants were mapped according to transmembrane helical orientation, while CALR exon 9 variants were evaluated according to predicted C-terminal charge profiles. Eight patients (4.5%) harboured atypical molecular signatures, including non-canonical MPL and CALR variants. MPL V501A mapped to the same transmembrane activation face as the canonical W515 hotspot, whereas MPL R592Q localized away from this region. Rare CALR 34-bp deletions generated positively charged C-terminal tails similar to canonical pathogenic frameshifts, while a 12-bp in-frame deletion preserved the native acidic profile despite high variant allele frequency. Atypical cases showed clinical overlap with canonical MPN cohorts and clustered within related phenotypic groups on unsupervised analysis. Rare MPL and CALR variants may share structural and electrostatic features with established MPN drivers. Integrating receptor structure, charge effects and clinical phenotype may help contextualize atypical variants detected on routine NGS panels.