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. 2022 Jun 23;18(6):e1010236. doi: 10.1371/journal.pgen.1010236

Fig 3. Rare variants in SMYD1 and BMP10 identified in family 346 are functionally damaging.

Fig 3

A. ES was performed on two BAV patients from family 346 (asterisks). Both individuals were heterozygous for the SMYD1 p.R441W and BMP10 p.R209C variants. B. In a luciferase assay, using a reporter construct driven by the SV40 promoter (pGL3-SV40), cotransfection of a SMYD1 WT expression construct resulted in a 50% reduction in luciferase activity, demonstrating that SMYD1 represses transcription from the SV40 promoter. Transfection of the SMYD1 p.R441W variant protein caused significantly greater repression (~70%) of the reporter activity, to the same level as that of the SMYD1 DCTD variant that lacks the C-terminal autoinhibitory domain of SMYD1. These results indicate that SMYD1 p.R441W is a gain-of-function variant. The data shown represent the mean of values from 3 independent experiments in which each of the samples was assayed in triplicate (error bars indicate standard deviation, **** p<0.0001, *** p<0.001). C. Western blots of whole cell extracts or filtered culture medium from HEK293T cells stably transfected with BMP10 WT or BMP10 p.R209C expression constructs showed reduced levels of both the cellular (prodomain) and secreted form (growth factor domain) of the p.R209C variant protein.