Continuing the discussion from Synonymous mutations in representative yeast genes are mostly strongly non-neutral:
A preprint went up on bioRxiv today responding to the claimed implications of the Shen study:
Synonymous mutations change the DNA sequence of a gene without affecting the amino acid sequence of the encoded protein. Although emerging evidence suggests that synonymous mutations can impact RNA splicing, translational efficiency, and mRNA stability1, studies in human genetics, mutagenesis screens, and other experiments and evolutionary analyses have repeatedly shown that most synonymous variants are neutral or only weakly deleterious, with some notable exceptions. In their recent article, Shen et al. claim to have disproved these well-established findings. They perform mutagenesis experiments in yeast and conclude that synonymous mutations frequently reduce fitness to the same extent as nonsynonymous mutations2 . Based on their findings, the authors state that their results “imply that synonymous mutations are nearly as important as nonsynonymous mutations in causing disease.” An accompanying News and Views argues that "revising our expectations about synonymous mutations should expand our view of the genetic underpinnings of human health"3. Considering potential technical concerns with these experiments4 and a large, coherent body of knowledge establishing the predominant neutrality of synonymous variants, we caution against interpreting this study in the context of human disease.
Simultaneously, a second preprint was released, this one analysing the specific methods and results of Shen et al:
In a recent paper, Shen et al. reported that most mutations in the coding regions of 21 yeast genes were strongly deleterious, and that the distributions of fitness effects were similar for synonymous and nonsynonymous mutations. Taken at face value, these results would conflict with well-established findings from a broad range of fields and approaches. Here, we argue that these results arose from a lack of appropriate controls for the impacts of background genetic effects in edited strains. A re-examination of the data in Shen et al. strongly suggests that it is entirely consistent with the expectation that most nonsynonymous and nearly all synonymous mutations have no detectable effects on fitness. We present analyses which show that the data is inconsistent with the proposed explanation that pervasive fitness effects of synonymous mutations arise from their effects on mRNA levels, that the sequence-based fitness assay overestimates fitness effects compared to the growth-based fitness assay, and that the observed wide fitness distributions for nonsense mutations are consistent with ‘off-target’ effects or other uncontrolled sources of biological variation contributing to measured fitness. We conclude by discussing the essential controls and other experimental design considerations that are required to produce interpretable results regarding the fitness effects of mutations in large-scale screens.