Abstract Around birth, red blood cell (RBC) β-like globin expression transitions from γ-globin (HBG1/HBG2; HBG) to β-globin (HBB), resulting in a switch from fetal hemoglobin (HbF, α₂γ₂) to adult hemoglobin (HbA, α₂β₂). This developmental switch underlies the postnatal onset of β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia, caused by mutations in HBB. In hereditary persistence of fetal hemoglobin (HPFH), rare deletions in the β-globin gene cluster sustain high HbF levels into adulthood, ameliorating or eliminating disease symptoms. Genome editing strategies that mimic HPFH aim to reactivate HbF in patients with SCD or β-thalassemia, but their efficacy depends not only on total HbF levels but also on the proportion of RBCs expressing HbF. To better understand how different HPFH-like deletions influence γ-globin expression at the single-cell level, we performed single-cell RNA sequencing (scRNA-seq) of reticulocytes from individuals with compound heterozygosity for HbS and either the 85 kb HPFH1 deletion or the 35.8 kb Aγδβ⁰-thalassemia (Aγδβ-thal) deletion. Both deletions are associated with pancellular HbF expression but differ in average HbF levels and clinical severity. We studied three siblings with HbS/HPFH1 and two unrelated individuals with HbS/Aγδβ-thal. Clinical data, hematologic parameters, and HbF–expressing RBC (F-cell) fractions were collected during well visits and at the time of blood collection for scRNA-seq. Participants with HbS/HPFH1 exhibited no SCD symptoms, normal hemoglobin and reticulocyte counts, and no sickled RBCs on peripheral smears. Their average HbF level was 40.54±1.56%, and F-cell fractions approached 99%. In contrast, participants with HbS/Aγδβ-thal had HbF levels of 27.98±2.10%, with F-cell fractions of 86.2% and 90.5%. One experienced multiple episodes of acute chest syndrome and vaso-occlusive pain, while the other had a milder course. At the time of scRNA-seq, hemoglobin levels were normal or mildly reduced, and reticulocyte counts were within or slightly above normal range. HbF levels in hemolysates were 35.03±1.1% for HbS/HPFH1 individuals and 25.42% and 19.42% for the two HbS/Aγδβ-thal individuals. F-cell fractions by flow cytometry were 99±0.36% for HbS/HPFH1 and 77.9% and 93.4% for HbS/Aγδβ-thal. scRNA-seq of sorted reticulocytes revealed distinct HBG expression patterns. HbS/HPFH1 reticulocytes showed more uniform and higher HBG expression across cells, while HbS/Aγδβ-thal reticulocytes exhibited greater heterogeneity, with a substantial fraction expressing little or no HBG mRNA. To further explore the functional consequences of this heterogeneity, we examined RBC mechanical properties and sickling behavior under hypoxic stress using a microfluidic assay. RBCs from participant HbS/Aγδβ-thal.1 showed a greater tendency to sickle under reduced oxygen tension compared to those from HbS/HPFH1.1–3, despite similar oxygen saturation profiles. Consistent with this, blood smears prepared under hypoxia revealed sickled and morphologically abnormal RBCs in HbS/Aγδβ-thal.1 but not in HbS/HPFH1.1–3. No such differences were observed in smears prepared in ambient air. These findings suggest that clinical differences between these genotypes are not solely due to average HbF levels, but also to the distribution of γ-globin expression among individual erythroid cells. The more homogeneous expression in HbS/HPFH1 likely contributes to the absence of symptoms, whereas the heterogeneous expression in HbS/Aγδβ-thal may permit residual HbS polymerization and clinical manifestations. Our results highlight the importance of γ-globin expression heterogeneity in modulating disease severity in SCD. Even when HbF levels are relatively high, uneven distribution across RBCs can result in insufficient protection against HbS polymerization. These findings have implications for the design and evaluation of HbF–inducing therapies, emphasizing the need to assess not only bulk HbF levels but also the uniformity of expression at the single-cell level. Variability in clinical outcomes among individuals with similar genotypes further suggests the influence of additional genetic or environmental modifiers, underscoring the multifactorial nature of SCD pathophysiology.
Doerfler et al. (Mon,) studied this question.