Abstract
The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table.
Table 1. Number of subjects recruited to date 98 Number of subjects with iPSC lines established 56 Average number of iPSC lines per subject 3 (total of 158 lines generated) Quality control status of iPSC lines All lines are expanded and banked, mycoplasma free, express pluripotency markers Subjects with target cells differentiated (erythrocytes) 25
Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell.
Disclosures
No relevant conflicts of interest to declare.
In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases