The Association Between MYH13 And Aniridia And The Biological Function Of This Gene

Objective:MYH13 encodes myosin heavy chain My HC-eo isoform,which is an important subunit of the myosin molecule.In previous work,our group identified a missense mutation of MYH13 in a pedigree with aniridia.Based on this finding,we investigated the association between MYH13 mutation and anirida,and explored the biological function of this gene at the cell and animal level.Methods:The intracellular localization of My HC-eo was assayed by confocal microscopy.The effects of MYH13 mutation on cell proliferation and apoptosis were evaulated by MTS assay and flow cytometry,respectively.The expression of Myh13in mice was analyzed by q RT-PCR and western blotting.Myh13^Ser181Tyrknockin（KI）mice and Myh13 knockout（KO）mice were generated by using CRISPR/Cas9technology.Eyes and extraocular muscles were observed by hematoxylin and eosin（H&E）staining and transmission electron microscopy（TEM）.RNA-seq was used to detect the differential gene expression in extraoclualr muscles from mouse with or without Myh13 knockout.Results:My HC-eo was localized in the cytoplasm of cells.MYH13 p.Ser181Tyr mutation did not affect the intracellular localization of My HC-eo and yielded no effect on cell proliferation and apoptosis.In wild type（WT）mice,Myh13 was specifically expressed in extraocular muscle.Phenotypic analysis of Myh13^Ser181Tyrknockin mouse showed that there was no significant difference in the expression of Myh13 gene in the extraocular muscles among WT,Myh13^wt/mtand Myh13^mt/mtmouse.Genotypic ratio in the offsprings from the self-cross of Myh13^wt/mt mouse was consistent with Mendel’s law.Myh13^wt/mt and Myh13^mt/mt mouse displayed no ocular abnormalities.The extraocular muscles from Myh13^Ser181Tyr knockin mouse revealed no obvious morphological differences according to H&E staining.The extraocular muscles of Myh13^mt/mt mouse presented sarcoplasmic reticulum dilation,as observed by TEM.Phenotypic analysis of Myh13 knockout mouse showed that the expression of Myh13 was reduced in extraocular muscles in Myh13^+/-mouse compared to WT mouse and Myh13 was hardly expressed in Myh13^-/-mouse.Genotypic ratio in the offsprings from the self-cross of Myh13^+/-mouse was consistent with Mendel’s law.There were no obvious abnormalities in body weight and serum biochemical analysis of Myh13^+/-and Myh13^-/-mouse.Only a minority of Myh13^-/-mouse presented eye abnormalities such as dilated pupils and iris atrophy.The extraocular muscles from Myh13 knockout mouse showed no obvious morphological differences according to H&E staining.Lipid droplets were identifed in the extraocular muscles from Myh13^+/-and Myh13^-/-mouse,as observed by TEM.Besides,sarcoplasmic reticulum dilation was observed in the extraocular muscles from Myh13^-/-mouse.RNA-seq analysis identified that 31 genes were down-regulated and 2 genes were up-regulated in the extraocular muscles of Myh13^-/-mouse compared to WT mouse.Krt17,Krt14 and Dsp were associated with intermediate filaments,which were down-regulated in the extraocular muscles of Myh13^-/-mouse validated by q RT-PCR.Conclusions:Mutation or knockout of Myh13 causes microstructure abnormality of the extraocular muscle in mice.The knockout of Myh13 results in the down-regulation of the expression of the intermediate filament related genes in the extraocular muscle,which may have an influence on cytoskeletal structure.The MYH13 mutation may not be associated with aniridia and further investigation is needed.