Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations.

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.