Background: Until now, three different isoforms of nonmuscle myosin heavy chain II protein have been identified in vertebrates: II-A, II-B, II-C. Mutations in the MYH9 gene encoding for the NMHC II-A have been identified in a group of thrombocytopenia disorders distinguished by different combinations of pathologies, such as deafness, nephritis, cataracts, and Dohle-like leukocyte inclusions. To date, more than 20 single amino mutations in MYH9 have been reported in patients with the above syndromes, however, the phenotype-genotype correlation remains to be elucidated. The mouse protein, NMHC II-A, shows 98% identity with the human protein, suggesting that it could serve as an appropriate experimental model. Proof of a mutation-pathology connection may bring new diagnostic tools and new treatments as well as to help understand the molecular pathogenesis of the syndromes resulting from a mutation in NMHC II-A.? Results: For this purpose, we are generating mice with a mutation in exon 31 leading to a D1424N substitution. This substitution was identified in patients with May-Hegglin and Fechtner syndromes from different, unrelated families.? To produce the D1424N mutation, we introduced a change of two nucleotides, GAC to AAT, using the appropriate primers. A fragment of 3.1 kb, including exons 28-31 with the mutation in exon 31, was inserted into a modified plasmid, pNTKV-LoxP, 5? to the gene encoding neomycin resistance and an additional sequence of 3.9 kb, including exons 32-38, was inserted 3? to the neomycin resistance gene. The nucleotide sequence of the targeting construct has been verified by sequencing and the purified plasmid will be transfected into ES cells.
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