Resolving mechanisms of immune-mediated disease in primary CD4 T cells

Abstract

Deriving mechanisms of immune-mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by linkage disequilibrium. To determine whether causal variants could be identified via their functional effects, we adapted a massively-parallel reporter assay for use in primary CD4 T-cells, key effectors of many immune-mediated diseases. Using the results to guide further study, we provide a generalisable framework for resolving disease mechanisms from non-coding associations -illustrated by a locus linked to 6 immune-mediated diseases, where the lead functional variant causally disrupts a super-enhancer within an NF-κB-driven regulatory circuit, triggering unrestrained T-cell activation. Keywords MPRA, primary T cells, TNFAIP3, super-enhancer, GWAS 7Shan, X. et al. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 20, 6945-57 (2000).

Functional genomics in autoimmune diseases

“…luciferase) assays are used to assess the regulatory impact of candidate DNA regions, such as those harboring candidate SNPs. This can be performed in a high-throughput manner as part of massively parallel reporter assays (MPRAs) and has recently been optimized for use in primary T-helper cells ( 52 ). This technique was applied to 14 autoimmune loci, in rested and stimulated primary T-helper cells, identifying SNPs which had the greatest impact on regulatory activity.…”

“…In the aforementioned study, Cas9 was targeted to multiple locations within close proximity of rs6927172 in primary T-helper cells, giving rise to a heterogeneous combination of deletions and other edits. Edited cells had reduced TNFAIP3 expression, with no change in five other neighboring genes ( 52 ). A similar approach was applied separately in HEK293 cells, generating clonally derived lines with specific deletions of 11/12 bp including rs6927172.…”

“Stripe” transcription factors provide accessibility to co-binding partners in mammalian genomes

“…The importance of these activities is highlighted by the fact that more than 80% of GWAS-identified polymorphisms associated with disease fall within regulatory elements (Giral et al, 2018;Maurano et al, 2012;Visel et al, 2009). To understand how such non-coding variants cause disease (a primary goal of translational research), several strategies are currently being implemented, including single-nucleotide polymorphism (SNP) enrichment methods (Cano-Gamez and Trynka, 2020), the statistical colocalization of variants to eQTLs (Giambartolomei et al, 2014), and genome-wide CRISPR screens (Bourges et al, 2020;Kampmann, 2020). Although these techniques are powerful, a full understanding of the variant-to-function problem will require the deciphering of the cis-regulatory code (Jindal and Farley, 2021).…”

Transcriptional-Regulatory Convergence Across Functional MDD Risk Variants Identified by Massively Parallel Reporter Assays

“…Delivery of a library of DNA elements to cells, followed by RNA collection and sequencing, enables quantitative estimation of the expression driven by each element as a ratio of collected RNA barcode to delivered DNA barcode. These assays have recently been adapted to systematically identify SNPs with functional allelic TR differences from GWAS loci for several diseases [48][49][50][51][52][53][54][55][56] . Two key features make MPRAs advantageous for identifying both functional SNPs and their TR interactions.…”