In the present study, we report results of a GWAS for 6 RBC traits (HGB, HCT, RBC count, MCV, MCH, and MCHC) in 12,486 patients of European ancestry from 5 sites that comprise the eMERGE network.9,38
We identified 15 chromosomal loci associated with at least one RBC trait, including 11 loci associated with RBC traits in prior cohort studies.4,5,7
Notably, genes present in 3 of the 4 loci newly identified in individuals of European ancestry (THRB
) and in 6 of the 11 replicated loci (KLF1
) are implicated in erythroid differentiation and regulation of cell cycle in hematopoietic stem cells. Our report also highlights the potential use of the EMR in conducting GWAS of quantitative traits of medical importance.
The sample size in this study was similar to that of previous GWAS of RBC traits in individuals of European ancestry,4,5
yet we were able to identify 4 new loci and replicate 11 loci that had been identified in at least 2 of the previous reports. Of the 4 new loci that were novel for individuals of European ancestry, 3 have been reported to be associated with RBC traits in Japanese individuals.8
An advantage of EMR-based GWAS of medically relevant quantitative traits is that the traits are measured by well-validated and established methods. In addition, multiple measures over time are often available, and these may permit study of the genetic basis of temporal trends in the traits. On the other hand, trait values may be affected by acute illness or chronic comorbid conditions present at the time of measurement, and appropriate EMR-based phenotyping algorithms for excluding such trait values need to be developed (as was done for this study) before genetic analyses.
In the present study, genes (PTPLAD1
, and CDT1
) residing in 3 newly identified loci associated with RBC traits are implicated in erythroid differentiation and cell cycle regulation (). Coordinated regulation of cell cycle progression and differentiation is critical for normal hematopoiesis. Cells committed to differentiation undergo a programmed loss of proliferative capacity, restricted to only a few divisions because of their irreversible growth arrest in G1
phase, and terminal erythroid differentiation is accompanied by arrest of the cell cycle in the G1
We briefly describe the role of these genes in cell proliferation and differentiation below.
FIGURE 3 Genes implicated in erythroid differentiation are associated with red blood cell (RBC) traits. Three novel genes (THRB, PTPLAD1, CDT1) shaded in blue and 6 replicated loci (KLF1, ALDH8A1, SPTA1, FBXO7, CCND3, TFR2/EPO) shaded in brown are shown. A, Different (more ...) PTPLAD1
potentiates Rac1-induced NF-κB and c-Jun N-terminal kinase activation and also forms complexes with constitutively activated Rac1.42
Rac1 (and Rac2) GTPases are necessary for early erythropoietic expansion in the bone marrow, and erythropoiesis in Rac1−/−
mice is characterized by abnormal burst-forming units–erythroid colony morphologic findings and decreased numbers of megakaryocyte-erythrocyte progenitors, colony-forming units–erythroid, and erythroblasts in the bone marrow.21
is also known as erythroblastic leukemia viral (v-erbA) oncogene homolog 2, avian. The v-erbA oncogene of avian erythroblastosis virus encodes an aberrant version of a gene for a thyroid hormone receptor (c-erbA) and promotes neoplasia by blocking erythroid differentiation. Ligand-activated c-erbA/TR accelerates erythroid differentiation, whereas unliganded c-erbA/TR effectively blocks erythroid differentiation.43
Expression of THRB
may vary according to cell cycle, thereby mediating hormone sensitivity and contributing to cell cycle progression during normal development.44
Cdk6 has been implicated in the terminal hematopoietic differentiation processes in mice, and loss of Cdk6 affects the production of terminally differentiated myeloid and erythroid cells.45
, which is required for the initiation of DNA replication, together with origin recognition complex and CDC6, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G1
In mice, CDT1 is phosphorylated by CDKs during the cell cycle, which induces the association of CDT1 with ubiquitination complex SCF-SKP2 and targets CDT1 for degradation.
Of the genes residing in the replicated loci, 6 were also involved in erythrocyte differentiation and regulation of cell cycle. Erythropoiesis is largely mediated by a relatively small number of lineage-restricted transcription factors, including KLF1
controls the development and differentiation of erythroid lineage by mediating the switch from expression of fetal γ-globin to adult β-globin and regulates transcription of genes that encode cytoskeletal proteins, heme synthesis enzymes, and blood group antigens.48,49
also regulates the lineage progression of megakaryocyte-erythroid progenitor cells. Failure of terminal erythroid differentiation in KLF1
-deficient mice is associated with cell cycle perturbation and reduced expression of E2F2.49
In humans, haploinsufficiency for KLF1
causes hereditary persistence of fetal hemoglobin.50
In erythrocyte progenitor cells, KLF1
was upregulated 28-fold when compared with hematopoietic stem cells.22
converts 9-cis-retinaldehyde into the 9-cis-retinoic acid, which influences hematopoiesis in the fetal liver.31
CCND3 interacts with CDK4 and CDK6 to play a lineage-independent role in hematopoiesis,51
SPTA1 and TFR2 are proteins related to erythrocyte structure and function, EPO is a hematopoietic growth factor, and EPOR is the required receptor. In the mouse, FBXO7 associates specifically with CDK6 to promote CDK6–cyclin D complex formation and cellular transformation.52
The effect of trait-associated alleles may be exerted by multiple mechanisms influencing gene expression.16
Of the SNPs associated with RBC traits in the present study, 15 SNPs in 7 loci were associated with 27 distant eQTLs (Supplemental Table 5
, available online at http://www.mayoclinicproceedings.org
). The associated SNPs may also influence epigenetic regulation, including methylation and histone modification.
Three of the 4 newly discovered loci associated with RBC traits in our analyses were reported in a Japanese cohort.8
Nonetheless, these loci are novel for individuals of European ancestry. The associations of these loci with RBC traits are weaker than the replicated loci, as expected. Our annotation of likely candidate genes and their function is based on LD patterns and known literature and bioinformatics analyses. Additional work is needed to confirm the role of specific genes at a locus in RBC biology.