Identification of Novel Variants Associated with Fetal Hemoglobin Levels in Healthy Donors (the INTERVAL study)

Sickle cell disease (SCD) affects more than five million people worldwide, predominantly in sub-Saharan Africa. Hereditary persistence of fetal hemoglobin (HbF) is an uncommon genetic condition in which production of HbF in early life is not suppressed. SCD symptoms are reduced in patients carrying this condition, suggesting that increased HbF levels may be a promising therapeutic strategy to ameliorate the symptoms of SCD. This is exemplified by the HbF-raising compound hydroxyurea, currently the most commonly used US Food and Drug Administration approved drug treatment for SCD. However, hydroxyurea is only effective in ~70% of patients and carries a black box warning for carcinogenicity, hence there is a need for additional SCD treatments.

Previous genome-wide association studies (GWAS) have identified four loci robustly associated with HbF levels, including variants in the BCL11A region. These initial genetic discoveries have led to promising ex vivo gene-editing approaches to silence or reduce levels of BCL11A, which are currently being tested in clinical trials. To identify novel potential therapeutic targets to raise HbF levels, we conducted the largest GWAS of HbF levels in ~11,000 healthy blood donors from the INTERVAL study among whom HbF was measured in whole blood using a mass spectrometry approach. We ran linear mixed models accounting for age, sex, blood group, technical effects and 10 principal components of ancestry for 14,910,742 variants either directly genotyped using the Affymetrix UK Biobank array or imputed from a combined 1000 Genomes/UK10K reference panel. In addition to confirming previously reported signals at the BCL11A, HBS1L-MYB and HBB loci, stepwise conditional analysis identified six novel genomic regions at genome-wide significance (p<5x10^-8). At some loci we identified multiple independent association signals, including two at BCL11A, three at HBS1L-MYB, and two at HBB. Genetic fine-mapping resolved some loci to highly likely causal variants (posterior probability>0.5), including a rare (frequency=0.2%) variant near GRIK2.

To identify likely causal genes and mechanisms, we integrated our results with relevant transcriptomic and epigenomic datasets. Preliminary results suggest that a common (frequency=26%) 29 base-pair indel upstream of CHAC2 is highly likely (posterior probability=0.77) to be the causal variant at this locus. The variant maps to a site of active chromatin and GATA1 transcription factor binding specifically in erythroblasts, and the insertion allele contains a GATA motif suggesting that this variant regulates HbF levels by the presence or absence of a second GATA1 binding site. Gene expression data across a range of hematopoietic cells revealed a restricted expression pattern of CHAC2 in erythroblasts, but not for other genes in the region, suggesting CHAC2 as the likely causal gene. CHAC2 encodes the glutathione-specific gamma-glutamylcyclotransferase 2, an enzyme that catalyzes the cleavage of glutathione into 5-oxo-L-proline and a Cys-Gly dipeptide. This finding adds support to the idea that altered erythrocyte glutathione levels play a role in SCD pathogenesis, potentially via HbF modulation. Ongoing work includes characterizing the likely causal mechanisms of the six novel loci, and providing target validation of CHAC2 using genome editing and deep phenotyping of erythroid cells.

In summary, our expanded GWAS has identified new loci associated with HbF levels, providing novel potential therapeutic targets for SCD.