Common α-globin variants modify hematologic and other clinical phenotypes in sickle cell trait and disease

Abstract

Co-inheritance of α-thalassemia has a significant protective effect on the severity of complications of sickle cell disease (SCD), including stroke. However, little information exists on the association and interactions for the common African ancestral α-thalassemia mutation (-α3.7 deletion) and β-globin traits (HbS trait [SCT] and HbC trait) on important clinical phenotypes such as red blood cell parameters, anemia, and chronic kidney disease (CKD). In a community-based cohort of 2,916 African Americans from the Jackson Heart Study, we confirmed the expected associations between SCT, HbC trait, and the -α3.7 deletion with lower mean corpuscular volume/mean corpuscular hemoglobin and higher red blood cell count and red cell distribution width. In addition to the recently recognized association of SCT with lower estimated glomerular filtration rate and glycated hemoglobin (HbA1c), we observed a novel association of the -α3.7 deletion with higher HbA1c levels. Co-inheritance of each additional copy of the -α3.7 deletion significantly lowered the risk of anemia and chronic kidney disease among individuals with SCT (P-interaction = 0.031 and 0.019, respectively). Furthermore, co-inheritance of a novel α-globin regulatory variant was associated with normalization of red cell parameters in individuals with the -α3.7 deletion and significantly negated the protective effect of α-thalassemia on stroke in 1,139 patients with sickle cell anemia from the Cooperative Study of Sickle Cell Disease (CSSCD) (P-interaction = 0.0049). Functional assays determined that rs11865131, located in the major alpha-globin enhancer MCS-R2, was the most likely causal variant. These findings suggest that common α- and β-globin variants interact to influence hematologic and clinical phenotypes in African Americans, with potential implications for risk-stratification and counseling of individuals with SCD and SCT.

Fig 1. Genomic context of the α-globin regulatory GWAS locus.

(A-B) The genomic region spanning the NPRL3 and HBA1 genes that contain the lead variant rs11248850 and its LD proxies, multi-species conserved sequences (MCS R1-4), and the -α3.7 deletion. eQTL(48, 49) and meQT(50) results from blood samples that reach genome-wide significance are shown, as well as the effect direction corresponding to the A alleles of rs11248850 and rs11865131. Although not illustrated, decreased expression of SNRNP2 was also associated in cis with the A allele. (B) rs11248850 and its LD proxies (magenta color indicates R² ≥ 0.9 and red indicates R² ≥ 0.8 but < 0.9). Signal tracks of DNaseI hypersensitivity(32), ChIP-seq for the key erythroid transcription factors GATA1 and TAL1(34), and histone modifications H3K4me1, H3K27ac and H3K4me3(33) from human derived erythroblasts are shown. The MCS-R2 enhancer element which overlaps rs11248850 and rs11865131 is highlighted in pink. (C) ChIP-seq occupancy sites are shown for transcription factors that were profiled in K562 cells and have motifs proximal to either of the MCS-R2 variants(43, 44). Evolutionary conservation across 100 vertebrates (PhyloP score)(47) and in silico mutagenesis(42) using a gkm-SVM trained on K562 DNase I hypersensitivity data identify likely functional motifs in the MCS-R2 element. Although rs11248850 appears to disrupt a GATA1 motif, it is not predicted by our software to have an effect as it resides in the low information content section between the GATA1 core and TAL1 core motifs.

Fig 2. Functional assays for α-globin enhancer variants.

Normalized activity (RNA barcode count divided by DNA barcode count) from the massively parallel reporter assay. The median of the entire library was set to 0; therefore an activity of 0 corresponds roughly to minP transcriptional levels. Only elements overlapping with the MCS-R2 element have robust regulatory activity in K562 cells. (B) Relative luciferase activity as compared to minP promoter for α-globin enhancer variants (rs11865131 and rs11248850). A significant allelic difference in enhancer activity is observed for rs11865131 (p = 6.91 x 10⁻³ for lower luciferase activity with A allele). (C) Allelic skew for rs11865131 from DNase I hypersensitivity (DHS) data in 46 heterozygous cell types previously identified in(37) and in erythroblasts only ***p<0.0001.