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In vitro expression and characterization of MYH9 mutant alleles linked to hereditary hearing loss
Calvin C Wei1, Anil K Lalwani, Anand N Mhatre
1Department of Otolaryngology, New York University School of Medicine, New York, NY 10016, USA.
Objective:
To assess whether MYH9 mutant alleles linked to hereditary hearing loss induce disruption of cellular functions and associated phenotype following transient expression within cultured human cell lines.
Study Design:
Dominantly inherited MYH9 mutant alleles, MYH9(R702C) and MYH9(R705H), were integrated within eukaryotic expression vector and then transfected into cultured human cell lines for transient expression and analysis. The transfected cells were assessed for transgene-induced alterations of the cellular phenotype, including NMHC-IIA-dependent cell shape, actin cytoskeleton integrity, and inhibition of cytokinesis.
Setting:
Laboratories of Molecular Otology and Molecular Genetics at the New York University School of Medicine.
Subjects And Methods:
HeLa and MDA-MB-231 cultured cell lines were transiently transfected with an expression vector carrying a wild type or mutant MYH9 alleles, linked to nonsyndromic and syndromic hearing loss. Expression of exogenous transgene product was detected with antibodies directed toward its N-terminal HA tag, and transfection efficiency was greater than 95 percent. Host cells were characterized for cell shape, integrity of actin-myosin cytoskeleton, and nuclear status before and after transfections via immunofluorescence.
Results:
MDA-MB-231 cells transfected with MYH9(R705H) but not MYH9(R702C) were found to have a greater than two-fold increase in cells with filopodia and a ten-fold increase in proportion of cells with multiple nuclei, indicating inhibition of cytokinesis, relative to the control cells transfected with wild type MYH9. Actin cytoskeleton configuration within MDA-MB-231 cells was unaffected by expression of MYH9(R702C) or MYH9(R705H). Unlike MDA-MB-231 cells, HeLa cells were refractory to MYH9(R705H) and MYH9(R702C).
Conclusions:
MYH9(R705H)-induced altered phenotype of the MDA-MB-231 cell line supports the pathogenicity of the mutation and represents a suitable assay system for identification and characterization of its dysfunction.
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