Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits

Abstract

Summary Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneous screen 2756 variants in strong linkage-disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164 using targeted genome editing. Functional follow up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type specific transcriptional regulatory pathways.

GATA1 erythroid-specific regulation of SEC23B expression and its implication in the pathogenesis of congenital dyserythropoietic anemia type II

“…It is likely that common genetic variants associated with small changes in erythrocyte production act by slightly altering the binding and activity of GATA1 and co-factors, resulting in mild to moderate changes in the expression of target genes. 15 In the study herein we demonstrated that the clinical features of the case B-II.2 are the result of a complex genotype SEC23B-GATA1, suggesting a novel genetic etiology underlying CDAII. We described a GATA1 polymorphism as a genetic modifier, which exacerbates the phenotype induced by mutations in GATA1-dependent genes.…”

Variant Interpretation: Functional Assays to the Rescue

“…58 MPRAs have also been used to simultaneously test thousands of variants associated with eQTLs 22 or variants in linkage disequilibrium with lead SNPs from GWASs for red blood cell traits. 59 Another noteworthy adaptation of MPRAs is population-scale self-transcribing active regulatory region sequencing (POPSTARR), in which candidate regulatory elements from numerous individuals are cloned via DNA sequence capture and tested in parallel. 60 However, regulatory grammar is not fully understood, and MPRAs take transcriptional regulatory elements out of their genomic and native chromatin context.…”

Quantifying the regulatory effect size of cis-acting genetic variation using allelic fold change

“…For disease-associated eQTLs, understanding the relationship between the quantitative expression effect in the cells and disease risk will be important for understanding molecular mediators of disease risk. Finally, the recent development of experimental approaches such as MPRA (Tewhey et al 2016;Ulirsch et al 2016), STARR-seq (Arnold et al 2013;Vockley et al 2015), and CRISPR genome editing assays (Canver et al 2015;Wright and Sanjana 2016) has created demand for translating summary statistics of eQTL mapping to quantifications that are interpretable as reflecting molecular events in the cell. Our biologically interpretable estimates of cis-eQTL effect sizes from population data can be directly compared with in vitro quantification of regulatory variant effects.…”

“…Estimating the relative effect of eQTL alleles on expression levels has applications in computational functional genetics analysis, as well as in analysis of genetic regulatory variants by experimental assays such as genome editing (Arnold et al 2013;Canver et al 2015;Vockley et al 2015;Tewhey et al 2016;Ulirsch et al 2016;Wright and Sanjana 2016). However, thus far there has been no consensus definition for eQTL effect size, with each study defining its own measure for quantifying regulatory effect size.…”