Quantitative determination of SLC2A1 variant functional effects in GLUT1 deficiency syndrome.

OBJECTIVE: The goal of this study is to demonstrate the utility of a growth assay to quantify the functional impact of single nucleotide variants (SNVs) in SLC2A1, the gene responsible for Glut1DS. METHODS: The functional impact of 40 SNVs in SLC2A1 was quantitatively determined in HAP1 cells in which SLC2A1 is required for growth. Donor libraries were introduced into the endogenous SLC2A1 gene in HAP1-Lig4KO cells using CRISPR/Cas9. Cell populations were harvested and sequenced to quantify the effect of variants on growth and generate a functional score. Quantitative functional scores were compared to 3-OMG uptake, SLC2A1 cell surface expression, CADD score, and clinical data, including CSF/blood glucose ratio. RESULTS: Nonsense variants (N = 3) were reduced in cell culture over time resulting in negative scores (mean score: -1.15 ± 0.17), whereas synonymous variants (N = 10) were not depleted (mean score: 0.25 ± 0.12) (P < 2e-16). Missense variants (N = 27) yielded a range of functional scores including slightly negative scores, supporting a partial function and intermediate phenotype. Several variants with normal results on either cell surface expression (p.N34S and p.W65R) or 3-OMG uptake (p.W65R) had negative functional scores. There is a moderate but significant correlation between our functional scores and CADD scores. INTERPRETATION: Cell growth is useful to quantitatively determine the functional effects of SLC2A1 variants. Nonsense variants were reliably distinguished from benign variants in this in vitro functional assay. For facilitating early diagnosis and therapeutic intervention, future work is needed to determine the functional effect of every possible variant in SLC2A1.

Comparative Transcriptomics Provides Insights into Reticulate and Adaptive Evolution of a Butterfly Radiation.

Butterfly eyes are complex organs that are composed of a diversity of proteins and they play a central role in visual signaling and ultimately, speciation, and adaptation. Here, we utilized the whole eye transcriptome to obtain a more holistic view of the evolution of the butterfly eye while accounting for speciation events that co-occur with ancient hybridization. We sequenced and assembled transcriptomes from adult female eyes of eight species representing all major clades of the Heliconius genus and an additional outgroup species, Dryas iulia. We identified 4,042 orthologous genes shared across all transcriptome data sets and constructed a transcriptome-wide phylogeny, which revealed topological discordance with the mitochondrial phylogenetic tree in the Heliconius pupal mating clade. We then estimated introgression among lineages using additional genome data and found evidence for ancient hybridization leading to the common ancestor of Heliconius hortense and Heliconius clysonymus. We estimated the Ka/Ks ratio for each orthologous cluster and performed further tests to demonstrate genes showing evidence of adaptive protein evolution. Furthermore, we characterized patterns of expression for a subset of these positively selected orthologs using qRT-PCR. Taken together, we identified candidate eye genes that show signatures of adaptive molecular evolution and provide evidence of their expression divergence between species, tissues, and sexes. Our results demonstrate: 1) greater evolutionary changes in younger Heliconius lineages, that is, more positively selected genes in the cydno-melpomene-hecale group as opposed to the sara-hortense-erato group, and 2) suggest an ancient hybridization leading to speciation among Heliconius pupal-mating species.

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.