Highlights: [•] Synonymous mutations, which do not result in a change in amino acid sequence, have been considered biologically silent. [•] Studies in molecular evolution show that synonymous codons are under selective pressure. [•] Synonymous mutation may affect the function of translated protein through diverse cellular mechanisms. [•] Multiple methods should be applied to investigate synonymous variants and decipher the exact mechanisms by which they impact biological function. [ABSTRACT FROM AUTHOR]