Clinical Analysis And Pathogenic Mechanism Of MYH9-related Disease

MYH9related disease (MYH9-related disease, MYH9-RD) is an autosomal dominantinherited disease, which is caused by mutation of non-muscle myosin heavy chain9gene(MYH9) located on human chromosome22q12.3. MYH9gene encodes non-musclemyosin heavy chain IIA (NMMHC-IIA) with molecular weight being about220kDa,which plays important roles in cell migration, cytokinesis, cell adhesion, cell morphology,cell polarity formation and cell tropism. However, MYH9-RD pathogenesis andNMMHC-IIA function are not yet explained clearly. Therefore, we investigated clinicaland genetic features of17patients with MYH9-RD; observed the effects of mutatedNMMHC-IIA on cell morphology; identified the NMMHC-IIA protein interactome;anlyzed the impact of NMMHC-IIA mutant on the proteome profiles. These studies mightbe in favor of clarifying NMMHC-IIA function and pathogenesis of MYH9-RD.Aim：1.Clinical features and genetic diagnosis of17patients with MYH9-RD;2.Preparation offive expression vectors of different NMMHC-IIA mutations (K74E, D1424N, W33R,E1841K and R702H-IIA), establishment of stable transfected HEK293cell lines, andobservation of inclusion bodies and morphology;3. Identification of NMMHC-IIAinteracting proteins;4. Analysis of proteome profiles by mutated NMMHC-IIA.Method：1. Hemocytometer and manual method for platelet count; Wright’s staining andimmunofluorescence staining for observation of platelet morphology and neutrophilinclusion bodies; extraction of DNA from peripheral blood of MYH9-related diseasepatients, amplification of40exons and flanking sequences at both ends using PCR, and DNA sequencing to determine genetic abnormalities; exclusion of polymorphism usingrestriction fragment length polymorphism analysis.2.Preparation of pEGFP-C3-NMMHC-IIA expression vector; using site-directedmutagenesis technique, PCR amplification of plasmid with K74E、D1424N、W33R、E1841K and R702H primers; Digested by DpnI and transformed into DH5α competentcells; Choosing and sequencing positive clones plasmid; Extraction of plasmid with highquality and high concentrations; transfection of these plasmids into HEK293cells byLipofectamine2000; Screening by G418; Observation of cell morphology andNMMHC-IIA inclusion bodies using immunofluorescence analysis.3. High-throughput identification of interacting protein of non-muscle myosin IIA usingco-immunoprecipitation technique and high performance liquid chromatography-tandemmass spectrometry method; Gominer analysis of non-muscle myosin IIA interactingproteins in molecular function, subcellular localization and biological processes.4. Proteome profiling of the impact of mutanted non-muscle myosin IIA using stableisotope-labeled amino acid cell culture (SILAC) quantitative proteomics technology;Gominer analysis of the distribution of molecular function of those changed proteins;Verifying the level of the above changed proteins using western blot technique.Results：1. With Wright’s staining the macrothrombocytopenia, light blue neutrophil inclusionbodies were observerd in14of17patients; small or faint inclusion bodies were observedin other3patients using immunofluorescence analysis, which were not found withWright’sstaining; Nine mutations of W33R, p.Q1443_K1445dup, K74E, D1447A, IVS25+1T→A,R702H, D1424N, R1933X, and E1945X were found, and the former five were firstlydiscovered;2. Five mutated pEGFP-C3-IIA vectors with mutation of K74E, D1424N, W33R, E1841Kand R702H-IIA were constructed; HEK293stable transfected cell lines were established;Inclusion bodies in HEK293cells except for overexpressed R702H were observed; thechanges of cytoskeleton morphological in those cell lines were different. 3. A total of4591peptides were identified, corresponding to151proteins interacting withNMMHC-IIA, and there is a very wide distribution of those proteins in molecular function,subcellular localization, and biological processes.4. Compared with the cells of overexpressed wild-type non-muscle myosin IIA, there weresignificant changes of the expression levels of92proteins in the cells of overexpressedmutated non-muscle myosin IIA. Among these, the levels of37proteins were remarkablyincreased, and the levels of55proteins were significantly decreased. These proteins had awide range of functional distribution; the expression levels of heat shock protein70(HSP70) and non-muscular myosin IIB were consistent with the results of large-scalequantitative proteomics analysis with Western Blotting.Conclusions：1.17cases of patients with MYH9-RD are confirmed, and nine mutations of which W33Rp.Q1443_K1445dup, K74E, D1447A, the IVS25+1T→A are firstly found.2. Construction the mutant plasmids of K74E, D1424N W33R, E1841K and R702H-IIA;After transfection and screening with G418, the HEK293stable cell lines overexpressingthese plasmids have been established; Except for R702H, inclusion bodies have beenobserved, and cells cytoskeletal have differently changed.3. The first database of NMMHC-IIA interacting proteins which contains151proteins wasestablished.4. The downregulation of55proteins, and the upregulation of37proteins caused byNMMHC-IIA mutant were identified, which are in favor of in-depth pathogenicmechanism studies for MYH9-related disease.