Major depressive disorder (MDD), one of the most frequently encountered forms of mental illness and a leading cause of disability worldwide1, poses a major challenge to genetic analysis. To date no robustly replicated genetic loci have been identified 2, despite analysis of more than 9,000 cases3. Using low coverage genome sequence of 5,303 Chinese women with recurrent MDD selected to reduce phenotypic heterogeneity, and 5,337 controls screened to exclude MDD, we identified and replicated two genome-wide significant loci contributing to risk of MDD on chromosome 10: one near the SIRT1 gene (P-value = 2.53×10−10) the other in an intron of the LHPP gene (P = 6.45×10−12). Analysis of 4,509 cases with a severe subtype of MDD, melancholia, yielded an increased genetic signal at the SIRT1 locus. We attribute our success to the recruitment of relatively homogeneous cases with severe illness.
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.
Alternative splicing (AS) generates isoform diversity for cellular identity and homeostasis in multicellular life. Although AS variation has been observed among single cells, little is known about the biological or evolutionary significance of such variation. We developed Expedition, a computational framework consisting of outrigger, a de novo splice graph transversal algorithm to detect AS; anchor, a Bayesian approach to assign modalities and bonvoyage, a visualization tool using non-negative matrix factorization to display modality changes. Applying Expedition to single pluripotent stem cells undergoing neuronal differentiation, we discover that up to 20% of AS exons exhibit bimodality. Bimodal exons are flanked by more conserved intronic sequences harboring distinct cis-regulatory motifs, constitute much of cell-type specific splicing, are highly dynamic during cellular transitions, preserve reading frame and reveal intricacy of cell states invisible to conventional gene expression analysis. Systematic AS characterization in single cells redefines our understanding of AS complexity in cell biology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.