Hemoglobin F mitigation of sickle cell complications decreases with aging
Authors: Seda S. Tolu, Morayma Reyes‐Gil, Ugochi O. Ogu, Merin Thomas, Eric E. Bouhassira, Caterina P. Minniti
Journal: American Journal of Hematology (2020)

To the Editor: Hemoglobin F (HbF) concentration is the major genetic modifier of clinical expression in sickle cell disease (SCD), and high levels improve survival by decreasing intracellular polymerization of hemoglobin S, theoretically affecting downstream vaso-occlusive and hemolytic complications (VOCs).1 However, total HbF level alone is not an accurate predictor of clinical complications as disease manifestations have been observed in individuals with the Arab-Indian haplotype.2 Distribution of HbF (pancellular or heterocellular) and the total amount of HbF/F per cell, as measured in picograms, impacts the ability of HbF to inhibit HbS polymerization. Hereditary persistence of fetal hemoglobin (HPFH) results in inherited HbF levels of ~30% (about 10 pg of HbF/F cell) that are sustained into adulthood. Those with deletional HPFH exhibit decreased or absent β-globin synthesis with pancellular distribution of HbF. Nondeletional HPFH, includes a variety of disorders resulting from mutations within the γ-globin gene, with either pancellular or heterocellular distribution of HbF.3 High levels of HbF are also induced by pharmacological methods, as with hydroxyurea (HU), resulting in heterocellular distribution.4 Individuals with HbS-HPFH have a relatively benign clinical course attributed to pancellular distribution of high HbF. Recent work by Steinberg et al hypothesized that if total HbF levels reach 30% with pancellular distribution, phenotypic cure of SCD should be achieved.5 As survival for patients with SCD has increased dramatically in the past 20 years, we hypothesized that SCD-related complications may increase with age, and progressively affect "high F" individuals. There is a concern that in everyday practice individuals with SCD and "high F," who typically have few vasocclusive crises in youth, are not further evaluated for the genetic mutation responsible for HPFH. And, they are often classified as "benign," leading to possible oversight of potential SCD related complications. Here we report the clinical characteristics of adult patients with SCD and inherited high HbF across different ages. Electronic medical records were reviewed for 769 patients with SCD, HbSS/SC/SB0/B+, at Montefiore Medical Center in the Bronx, New York. HbF values using high performance liquid chromatography (BioRad Variant II). Values obtained immediately after a blood transfusion were excluded. A comprehensive chart review for patients with HbF > 14% was completed for VOCs, opioid therapy, acute chest syndrome (ACS), avascular necrosis (AVN), splenomegaly or infarction (including evidence of calcification), pneumonia, retinopathy confirmed by ophthalmology report, stroke, pulmonary hypertension or elevation of tricuspid regurgitant jet velocity (TRV), leg ulcer, myocardial infarction (MI), priapism, chronic kidney disease (CKD with GFR <60), and miscarriage. HbF values were obtained using high performance liquid chromatography (BioRad Variant II). Clinical laboratory values were collected at steady state, or when patient was free of illness. For genetic analysis, all patients had beta globin mutation completed and 12/13 patients had alpha globin mutation completed. Since this cohort of patients sustained high lifetime HbF in the absence of HU, this prompted further analysis with flow cytometry and beta globin dosage analysis for HPFH. In 10/13 subjects, cellular distribution of HbF was analyzed by flow cytometry (CytoFlex, Beckman Coulter, CA) following fixation, permeabilization, and incubation of red blood cells with a monoclonal antibody against HbF, directly conjugated with FITC (Cat # MHFH01, Life Technologies, MD). Beta globin dosage analysis was performed in 7/13 patients using multiplex-polymerase chain reaction (PCR) amplification of fragments specific for portions of the epsilon-, gammaG-, gammaA-, delta-, and beta-globin genes. We identified 150 adults with SCD and HbF ≥ 14%; 137/150 of them, or 91% were taking HU, and were excluded from this analysis. Thirteen (9%) had ≥ HbF 14% and were either never prescribed HU, or received it for <6 months. This cohort comprises 10 women and three men, aged 23 to 72 years, 11 Black/African American, one Hispanic, and one Middle Eastern. Genotypes included 10 HbSS, five with concomitant alpha thalassemia trait, one with HbS/B0, and one HbS/HPFH. Note, HbF ranged from 14.6% to 30.1% with an average of 22.65% (SD ± 4.9). Figure 1A shows patients organized in ascending age, with review of relevant SCD-related complications. All patients experienced VOCs regardless of age or HbF level, 92% were prescribed opioids for SCD-related pain, 46% had AVN, 38% splenomegaly/infarction, 31% leg ulcers, 31% ACS, and 31% retinopathy. Laboratory data in supplementary Table S1 is notable for some indexes of hemolysis, mild degree of anemia and inflammation, with average hemoglobin of 9.66 g/dL (SD ± 1.52), reticulocyte 7.29% (SD ± 3.67), ferritin 470 ng/mL (SD ± 257.12). Flow cytometry revealed heterocellular distribution of HbF in 10 patients and pancellular distribution in patient two depicted in Figure 1B. Only patient two tested positive for the deletional variant of HPFH. Presence of non-deletional variants of HPFH remains unknown. When the number of complications per patient were plotted against age, a positive correlation was observed with R2 = 0.70 (P < .01).When number of complications was plotted against HbF, no correlation was observed with R2 = 0.006 (Figure S1). Here, we describe the clinical complication profile of 13 patients with SCD and inherited high HbF, age range 23 to 72 years. Clinical complications represented in Figure 1A shows that all patients experience varying degrees of SCD-related complications, despite maintenance of high HbF levels. Notably, all patients experienced VOCs, event patient two with pancellular HbF of nearly 30%.When the number of complications per patient were plotted against age, a positive correlation was observed with R2 = 0.70 (P < .01), while no correlation was observed when compared to HbF (R2 = 0.006, P < .01). This suggests that there is no threshold above which all complications are prevented, and SCD related complications occur with increasing age despite maintenance of high HbF. For example, patient two, a 25-year-old female with HbF ~30% and pancellular distribution, experiences emergency department visits 1-2 times per year for VOCs. She is on chronic opioid therapy for generalized joint pain. She displays minimal markers of inflammation and hemolysis (Hgb 11.7 g/dL, LDH 145 IU/L and total bilirubin 0.6 mg/dL). Her complication profile is relatively benign, and her pancellular distribution may offer her lifelong protection. In comparison, patient eight and 11, a male and female with lifetime HbF of close to 30% (29.3% and 30.1%, respectively), but with heterocellular distribution, suffered far more complications. Both patients experienced VOCs and were prescribed chronic opioid therapy for SCD-related pain. Patient 11 also experienced AVN of bilateral humeral heads, and retinopathy while patient eight suffered from recurrent leg ulcers and splenomegaly. Both have elevated markers of inflammation and hemolysis (Hgb 12.8 g/dL, and 9.4 g/dL, LDH 190 IU/L and 351 IU/L, total bilirubin 1.6 mg/dL and 1.5 mg/dL, respectively) when compared to patient two with pancellular distribution. These two patients despite lifetime HbF of ~ 30% seem to demonstrate a progressively worsening clinical profile with increasing age. We hypothesize that younger patients in this cohort with heterocellular distribution of HbF will likely develop a complication profile similar to that of patient 13, who had HbF of 26.1% with VOCs, AVN, splenic atrophy, ACS, pneumonia, coronary artery disease, MI, CKD, and death at age 72 from septic shock. Unfortunately, we do not have genetic analysis of this patient to confirm or disprove her HPFH status. The disease profile of SCD in high resources countries has changed dramatically over the past few decades as majority of patients now reach adulthood.6 Our report underscores that in patients with SCD and inherited high HbF, clinical outcomes depend on age and the distribution of HbF, and not merely on total HbF. Despite high lifetime HbF, these patients suffered SCD related vaso-occlusive and vasculopathic complications. There was no correlation between HbF levels and number of complications, suggesting that inherited high levels of HbF, even up to 30%, do not completely abrogate SCD related complications, but may only delay their onset and/or decrease their severity. A plausible explanation is that the distribution of HbF within erythrocytes was heterocellular within most of the patients studied, and many (12/13) did not sustain lifetime HbF of 30% or greater. Limitations of this report include inherent difficulty of identifying an accurate control group, as patients on HU would need to be prospectively analyzed for development of complications over time, especially before starting HU. Survival bias would make comparison difficult, as sicker patients may have died and an older cohort would be representative of individuals with milder disease. We conclude that strong vigilance should be maintained when treating an aging SCD population, and even those with high HbF may require multimodal therapy in order to achieve phenotypic cure. Measuring intracellular HbF distribution by flow cyotmetry maybe a useful biomarker when evaluating individuals with high HbF and assessing their risk level. Table S1. Clinical laboratory data for patients with Hb F > 14% in absence of hydroxyurea. Figure S1. Correlations with age and Hemoglobin F%. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.