Search for: All records

BackgroundUnderstanding genetic underpinnings of immune-mediated inflammatory diseases is crucial to improve treatments. Single-cell RNA sequencing (scRNA-seq) identifies cell states expanded in disease, but often overlooks genetic causality due to cost and small genotyping cohorts. Conversely, large genome-wide association studies (GWAS) are commonly accessible. MethodsWe present a 3-step robust benchmarking analysis of integrating GWAS and scRNA-seq to identify genetically relevant cell states and genes in inflammatory diseases. First, we applied and compared the results of three recent algorithms, based on pathways (scGWAS), single-cell disease scores (scDRS), or both (scPagwas), according to accuracy/sensitivity and interpretability. While previous studies focused on coarse cell types, we used disease-specific, fine-grained single-cell atlases (183,742 and 228,211 cells) and GWAS data (Ns of 97,173 and 45,975) for rheumatoid arthritis (RA) and ulcerative colitis (UC). Second, given the lack of scRNA-seq for many diseases with GWAS, we further tested the tools’ resolution limits by differentiating between similar diseases with only one fine-grained scRNA-seq atlas. Lastly, we provide a novel evaluation of noncoding SNP incorporation methods by testing which enabled the highest sensitivity/accuracy of known cell-state calls. ResultsWe first found that single-cell based tools scDRS and scPagwas called superior numbers of supported cell states that were overlooked by scGWAS. While scGWAS and scPagwas were advantageous for gene exploration, scDRS effectively accounted for batch effect and captured cellular heterogeneity of disease-relevance without single-cell genotyping. For noncoding SNP integration, we found a key trade-off between statistical power and confidence with positional (e.g. MAGMA) and non-positional approaches (e.g. chromatin-interaction, eQTL). Even when directly incorporating noncoding SNPs through 5’ scRNA-seq measures of regulatory elements, non disease-specific atlases gave misleading results by not containing disease-tissue specific transcriptomic patterns. Despite this criticality of tissue-specific scRNA-seq, we showed that scDRS enabled deconvolution of two similar diseases with a single fine-grained scRNA-seq atlas and separate GWAS. Indeed, we identified supported and novel genetic-phenotype linkages separating RA and ankylosing spondylitis, and UC and crohn’s disease. Overall, while noting evolving single-cell technologies, our study provides key findings for integrating expanding fine-grained scRNA-seq, GWAS, and noncoding SNP resources to unravel the complexities of inflammatory diseases. 

Regulation of gene expression is a critical link between genotype and phenotype explaining substantial heritable variation within species. However, we are only beginning to understand the ways that specific gene regulatory mechanisms contribute to adaptive divergence of populations. In plants, the post-transcriptional regulatory mechanism of alternative splicing (AS) plays an important role in both development and abiotic stress response, making it a compelling potential target of natural selection. AS allows organisms to generate multiple different transcripts/proteins from a single gene and thus may provide a source of evolutionary novelty. Here, we examine whether variation in alternative splicing and gene expression levels might contribute to adaptation and incipient speciation of dune-adapted prairie sunflowers in Great Sand Dunes National Park, Colorado, USA. We conducted a common garden experiment to assess transcriptomic variation among ecotypes and analyzed differential expression, differential splicing, and gene coexpression. We show that individual genes are strongly differentiated for both transcript level and alternative isoform proportions, even when grown in a common environment, and that gene coexpression networks are disrupted between ecotypes. Furthermore, we examined how genome-wide patterns of sequence divergence correspond to divergence in transcript levels and isoform proportions and find evidence for both cis and trans-regulation. Together, our results emphasize that alternative splicing has been an underappreciated mechanism providing source material for natural selection at short evolutionary time scales.