Congenital Macrothrombocytopenia and Defective Localization of the Nonmuscle Myosin Heavy Chain IIA in Leukocytes and Megakaryocytes with a Normal MYH9 Gene.

Autosomal dominant macrothrombocytopenias such as the May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome are all characterized by mutations in the MYH9 gene. It has been proposed that these MYH9-related diseases are not distinct entities but represent a variable expression of a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to more severe conditions encompassing hearing loss, cataracts and renal failure. Mutations in the MYH9 gene cause abnormal expression and/or function of the 224 kDa nonmuscle myosin heavy chain IIA (NMMHC-IIA) protein. Class II myosins are hexameric complexes composed of 2 heavy chains and 2 pairs of light chains forming a structure containing 2 N-terminal globular domains and an elongated α-helical C-terminal tail. Since identical mutations in the MYH9 gene cause variable clinical presentations in different patients, other factors likely modulate the mutant phenotype. We describe a patient with congenital macrothrombocytopenia, mild bleeding problems, Döhle-like leukocyte inclusion bodies, normal hearing, normal renal function and absent cataracts. The patient has macrothrombocytes with an average manual platelet count of 100 x 10⁹/L. Immunofluorescence confocal microscopy using an antibody specific for NMMHC-IIA revealed unique localization of NMMHC-IIA in the patient’s leukocytes. Platelets and leukocytes from both parents and one sister are normal. Megakaryocytes cultured from the patient’s and one parent’s peripheral blood CD34+ cells also demonstrated abnormal distribution of NMMHC-IIA in only the patient’s megakaryocytes. Ultrastructural analysis using electron microscopy revealed distinct inclusion bodies in the patient’s leukocytes and megakaryocytes that were not observed in either parent. The patient’s macrothrombocytopenia together with the abnormal distribution of NMMHC-IIA within leukocytes is highly suggestive of a MYH9-related disorder. The absence of these findings in either parent suggested a de novo MYH9 mutation. We therefore sought to identify the mutation via amplification and DNA sequencing of all MYH9 exons and intron/exon boundaries. Exons 1, 10, 16, 24, 25, 26, 30, 38 and 40 representing all of the previously described mutations in MYH9-related disorders were found to be normal. Surprisingly, the remaining MYH9 gene exons and intron/exon boundaries were also normal. This unexpected finding suggests that another factor is involved in the normal assembly and/or localization of NMMHC-IIA in human leukocytes and megakaryocytes. It also suggests that a deficiency of this factor may lead to congenital macrothrombocytopenia with features indistinguishable from those of MYH9-related disorders. Identification of this factor could enhance our understanding of NMMHC-IIA-related congenital macrothrombocytopenias and would allow us to gain insights into the normal assembly and/or function of NMMHC-IIA in human megakaryocytes and platelets.