Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis

Abstract

Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

Transcription Factor GFI1B in Health and Disease

“…Instead, hyperexpression and knockdown experiments in human primary cells have shown that the long GFI1B is required for megakaryopoiesis, but not the short form (25), which may have an inhibitory effect on platelet production (26) (Figure 2). …”

Discovery and refinement of genetic loci associated with cardiometabolic risk using dense imputation maps

“…GFI1B is a hematopoietic transcription factor required for normal red blood cell and platelet production 34. In a companion paper, we demonstrate that the rs150813342 variant influences the relative amounts of two GFI1B transcript isoforms, a full-length (long) and short isoform lacking the alternatively spliced exon 5 22. We further demonstrate the lineage-specific role of the long GFI1B isoform on megakaryocyte development.…”

“…SHROOM3 rs10008637 associated with higher PCV is an LD proxy (r 2 =0.98) for rs13146355, a common intronic variant associated with lower serum creatinine in East Asians 20 and higher serum magnesium in Europeans 21. One of the PLT-associated loci, synonymous variant rs150813342 of GFI1B , is reported concurrently in an independent exome sequencing data set 22.…”

Combined alpha-delta platelet storage pool deficiency is associated with mutations in GFI1B

“…In erythrocytes, the conditional inactivation of Gfi1b leads to impaired differentiation of pro-erythroblasts to mature erythrocytes [31]. A long isoform of GFI1B that contains exon 5 is essential for megakaryopoiesis, while a short isoform lacking exon 5 is essential for its erythropoietic role [14]. …”

“…In addition, a GFI1B mutation segregated with an isolated bleeding diathesis of variable severity in a family with mild to moderate macrothrombocytopenia and subtle red cell changes consisting of anisopoikylocytosis and increased red blood cell distribution width [10,11], and in a family with thrombocytopenia, enlarged hypogranular platelets and abnormally shaped red blood cells [12]. Indeed, GFI1B is known to participate in both megakaryopoiesis and erythropoiesis [13,14]. …”