The E1841K Mutation in MYH-9 of a Patient with May-Hegglin Anomaly Inhibits the Disassembly of Non-Muscle Myosin IIA (MYH-9) Responsible for the Phenotype of the Disease.

It has been demonstrated that MYH-9 (non-muscle myosin IIA) is responsible for the hereditary macrothrombocytopenia, such as May-Hegglin Anomaly (MHA), Fechtner syndrome (FS) and Sebastian syndrome (SS). We identified the E1841K mutation of MYH-9 gene of a patient with May-Hegglin Anomaly. Immunofluorescent staining of her peripheral blood smear samples revealed that non-muscle myosin IIA and actin filaments were co-localized at the Döhle-like inclusion bodies of neutrophils. To investigate the mechanism by which the E1841K mutation causes the phenotype of MHA, we first expressed GFP-tagged wild type and E1841K mutant MYH-9 cDNA in NIH3T3 cells. The mutant was able to form filaments and some inclusion bodies such as Döhle-like bodies, although it seemed to make no influences on the shape of the fibroblasts. Next we expressed the GFP-tagged wild and E1841K mutant MYH-9 in HEL cells and induced differentiation with TPA. HEL cells expressing the E1841K mutant failed in full differentiation. They could not produce the proplatelet-like projections as the cells overexpressing wild type of MYH-9 did. These results demonstrate that overexpression of this mutant can induce some phenotype similar to MHA. To clarify what molecular characteristics of the mutant myosin can cause the MHA phenotype, we expressed the recombinant wild and mutant proteins of non-muscle myosin IIA using Baculo-virus system. The mutant myosin failed to dissolve even in high ionic strength, suggesting this mutant can assemble in cytoplasm even under the condition, in which wild type myosin changes the conformation and exchanges dynamically assembly to disassembly. Next we induced myosin filaments in HEL cells by over-expressing the mutant of myosin light chain (DD mutant), which mimics the double-phosphorylated form. The DD mutant of myosin light chain also inhibited the proplatelet-like projections. Moreover silencing of MYH-9 gene with siRNA could have no inhibitory effects on the differentiation of HEL cells, producing rather more proplatelet-like projections than the control. These results indicated that filament formation of myosin is not critical for proplatelet-like projections, but disassembly of myosin is rather essential. Collectively, the E1841K mutant inhibits the disassembly of myosin to prevent the proplatelet-like formation of HEL cells, and the assembled myosin forms the Döhle-like inclusion bodies probably with actin filaments.