Study of Two Subjects with Absence of Severe Anemia with Homozygous β⁰ Thalassemia Due to Disrupted γ-to-β Switch

Sickle cell disease (SCD) and β thalassemia due to defects of the HBB (β globin gene) are among the most common inherited genetic disorders. At birth, there is a switch of γ globin transcription to β and d, with replacement of HbF by HbA and HbA2 virtually completed by six months of age. At that time, serious inherited disorders of the β gene, such as sickle cell disease and Cooley's anemia (homozygosity for β⁰ thalassemia mutations), become clinically apparent. Cooley's anemia is a life-threatening disorder wherein, in most patients, chronic transfusions or bone marrow transplantations are needed to sustain life. Rare patients with homozygosity or compound heterozygosity for β⁰ have no or only mild anemia. These patients maintain a high level of γ globin synthesis, apparently from a disrupted γ-to-β switch, thus attenuating their disease state. Recent work has demonstrated that BCL11A plays an important role in the suppression of γ-globin expression, as do polymorphisms of the gene that remain to be fully elucidated at a functional level. We recruited two unrelated subjects with homozygous β⁰ thalassemia mutations with no or only mild anemia (Patient #1, IVS2+1 G>A; Hb 14.2 Gm%; 97.2% HbF, 2.8% HbA₂, Patient #2, IVS 2 G-T; Hb 11.2 Gm%; 92/5% HbF, 6.8% HbA₂, 0.7% HbA). We sequenced transcripts and genomic loci of BCL11A from these patients. No mutations or splicing variants on transcripts were found. However, when the ≂f102 kb of genomic material from patient #1 was sequenced, 5 single nucleotide changes at intron II were found (2 known and 3 previously unpublished), while no genomic changes were found in patient #2. We then performed in vitro erythroid expansion from peripheral blood utilizing high erythropoietin concentrations and analyzed the cell proliferation and expression of globin and BCL11A genes. Interestingly, detectable amounts of β-globin transcripts were present in both patients during expansion, although protein levels were not detectable by the conventional HPLC method, probably due to limited sensitivity of this assay. Patient #1 showed mild in vitro induction of β-globin expression, which is lower than the control group, but no apparent cell proliferation. Patient #2 showed no induction of β-globin expression and hyperproliferation at a later stage of expansion (See Figure); however, the levels of BCL11A and γ-globin transcripts were indistinguishable from controls.