Abnormal subcellular localization of neutrophil nonmuscle myosin heavy chain-A (NMMHCA) is associated with MYH9 mutations in patients with autosomal dominant macrothrombocytopenia with leukocyte inclusions

Abstract number: P0752

Matsushita^* T., Kunishima† S., Kojima‡ T., Kamiya† T., Hirano§ M., Saito¶ H.

†Japanese Red Cross Aichi Blood Center, Japan; §Meijo University School of Pharmacy, Japan; ¶Nagoya National Hospital, Japan ‡Nagoya University School of Health Sciences, Japan; ^*Nagoya University School of Medicine, Japan;

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May–Hegglin anomaly (MHA), Sebastian syndrome (SBS), and Fechtner syndrome (FTNS), are rare disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Dohle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome (EPS) is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. We previously mapped the locus for these disorders on chromosome 22q12.3-q13.2 and found mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). Mutations have been found throughout the MYH9 from 27/30 patients and all were heterozygous, whereas the clear phenotype–genotype relationships were not found. Here, we developed unique immunofluorescence technique for conventional air-dried blood smears and studied the neutrophil NMMHCA localization from the 30 cases. Abnormal subcellular localization was observed in every neutrophil from 27 individuals harboring MYH9 mutations and the localization pattern were classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. By superimposing with the May–Grunwald-Giemsa stained glasses, NMMHCA coexisted with the neutrophil-inclusion bodies. In three cases, inclusion bodies were not found in conventional blood smears, but our technique clearly detected the abnormal NMMHCA localization and these patients had mutations in the MYH9. In contrast, other three cases with EPS or the isolated macrothrombocytopenia had normal NMMHCA localization and there were no MYH9 mutations. The antibody recognizing the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations at the C-terminus, suggesting that heterodimerization of normal and mutant NMMHCA results in the formation of inclusion bodies. These facts may interpret the dominant inheritance of MYH9 disorders and immunofluorescence analysis of neutrophil NMMHCA is a useful test to aid the clear hematopathologic classification of these disorders.