A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia

Abstract

Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) β-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34⁺ cells. As CD34⁺ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.

Figure 1. Association of rs4527238 in ANTXR1 with HbF expression in Saudi AI Cohort I (n=120), Saudi AI Cohort II (n=139), Indian AI (n=44), Saudi Benin (n=76) and CSSCD (n=894) datasets

The data for Saudi AI Cohort 1, Saudi Benin and CSSCD were based on genome-wide SNP analysis imputed to the 1000 Genomes reference panel while rs452728 was genotyped directly in the Indian AI cohort. The CSSCD sample included 47% Benin, 27% Benin/Bantu, 10% Benin/Senegal and 16% other haplotypes. The bar in each rectangle is the median value for the HbF in percentage scale

Figure 2. Immunofluorescence co-staining studies of ANTXR1 and HbF in sickle iPSCs

As ANTXR1 staining falls from culture days 15 to 20 and 26 (top row), HbF staining increases (bottom row). This reflects increased HbF synthesis as iPSCs mature and decreased HbF synthesis as CD34⁺ cells differentiate. A similar pattern was seen for the ɛ-globin chain (data not shown). Under our culture conditions established for erythroid differentiation of human CD34⁺ cells 80–90% of cells are erythrocytes on day 22, based on FACS analysis and Wright-Giemsa-staining (data not show). When the iPSCs were induced toward erythroid differentiation, >80% of cells are erythrocytes on day 15 of culture. Occasional bright ANTXR1 staining cells are non-erythroid based on Wright-Giemsa staining.