The current study should be interpreted in the context of the following limitations. First and foremost, our study used small RNA-seq samples that lacked direct replication or validation samples. Results from small samples may incur unreliable effect size estimates and could be more prone to a winner’s curse. But, we sought to assess the reproducibility of our findings by utilizing multiple tissue and data types as well as cross-referencing findings with GWASs that contain much larger samples. Polygenic scores and differential splicing associations with AUD are likely both associated with chronic alcohol use and thus may confound interpretations of pure genetic links with mRNA splicing in human brain tissue. Polygenic scores used PRScise2, which chooses a threshold that maximizes prediction and over-fits the data. Partitioned heritability analyses indicated that alternative mRNA splicing explained a significant amount of the heritability, but this is still ~ 2% of the total individual differences in AUD and may include non-splicing related DNA variants. The GWASs used in our study included some overlapping participants (e.g., UK BioBank and Million Veterans Project) and were limited to
