In The Blood: Connecting Variant to Function In Human Hematopoiesis

Abstract

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with a range of human diseases and traits. However, understanding the mechanisms by which these genetic variants have an impact on associated diseases and traits, often referred to as the variant-to-function (V2F) problem, remains a significant hurdle. Solving the V2F challenge requires us to identify causative genetic variants, relevant cell types/states, target genes, and mechanisms by which variants can cause diseases or alter phenotypic traits. We discuss emerging functional approaches that are being applied to tackle the V2F problem for blood cell traits, illuminating how human genetic variation can impact on key mechanisms in hematopoiesis, as well as highlighting future prospects for this nascent field.

Keywords: GWAS; blood cell traits; hematopoiesis; variant to function.

Figure 1.. Post- GWAS (genome-wide association study) workflow.

The figure represents a generalized process moving from studying variant to function in human hematopoiesis. Candidate GWAS loci are first dissected by fine-mapping to identify potential causal variants, which can be further prioritized with in silico prediction, functional annotation, and experimental validation methods. Once the target cell types in which these variants act are identified, one can perform experiments to define variants such as reporter assays, enhancer silencing or disruption (variant-centric), or perform experiments to identify genes such as loss-of-function screens (gene-centric). These approaches and additional functional studies can lead to the identification of mechanisms impacted by the genetic variant.

Figure 2.. Schematic of the human hematopoietic hierarchy.

Dashed lines indicate recently discovered differentiation paths. HSC, hematopoietic stem cell; MPP, multipotent progenitor; LMPP, lymphoid-primed multipotent progenitor; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte-erythroid progenitor; NK, natural killer cell; CD4, CD4⁺ T cell; CD8, CD8⁺ T cell; B, B cell; pDC, plasmacytoid dendritic cell; mono, monocyte; mDC, myeloid dendritic cell; gran, granulocyte; ery, erythroid; Mega, megakaryocyte. The looping arrow represents the self-renewal ability of hematopoietic stem cells. 16 blood traits that have been genetically studied are shown below the hierarchy.

Figure 3.. Scalable approaches for tackling the V2F challenge.

Displayed in the figure are some high-throughput approaches used to prioritize genetic variants identified from GWAS studies. The majority of GWAS nominated variants affect regulatory elements such as enhancers, thereby affecting gene expression. Effects of genetic variants on transcriptional activity of thousands of regulatory elements can be efficiently studied using pooled reporter constructs exogenously introduced into relevant cell types. Alternatively, pooled CRISPR/Cas9 screens can be applied to endogenously perturb either the regulatory element, target gene, or genetic variant in their native chromatin state. Various functional assays, such as single-cell RNA sequencing (e.g. as depicted by a gene expression heat map), cell proliferation/survival assays, and flow cytometry, can be used as functional readouts of CRISPR-Cas9 screens. UTR, untranslated region; barcodes, unique DNA sequences that identify reporter constructs; CRISPRi, CRISPR interference; CRISPRa, CRISPR activation. The methods listed are not comprehensive, but the figure depicts a general representation of the mechanistic progression involved in the V2F arc.