Clinical phenotypes of three children with sickle cell disease caused by <scp>HbS</scp>/Sicilian (δβ)<sup>0</sup>‐thalassemia deletion
Authors: Banu Aygun, Adriana Bello, Alexis A. Thompson, Lance Davis, Yanan Sun, Hong‐yuan Luo, Shuaiying Cui, David H. K. Chui
Journal: American Journal of Hematology (2022)

Between 10% and 15% of sickle cell disease (SCD) are caused by compound heterozygosity for sickle hemoglobin (HbS) and β-thalassemia mutations. Patients with HbS/β0-thalassemia usually have microcytic, hypochromic anemia with elevated HbA2 levels and are symptomatic. Their average HbF level is 8%, although it varies widely among patients. On the other hand, HbS/hereditary persistence of fetal hemoglobin (HPFH) exhibits no anemia or reticulocytosis, and HbF levels persist over 30%. Most of these patients are asymptomatic.1 Herein, we report three children with the unusual genotype of HbS/Sicilian (δβ)0-thalassemia deletion. Two of them suffered from SCD-associated complications in spite of markedly elevated HbF levels with pancellular distribution. Patient #1: This boy first presented to the hematology clinic at 2 months of age for newborn screening follow-up. His parents are of Asian Indian descent and nonconsanguineous. His father's blood counts are: Hb 13.9 g/dL; mean corpuscular volume (MCV) 68.5 fL; HbA 87.7%; HbA2 1.7%; and HbF 10.6%. His mother has sickle cell trait (HbA 61.2%; HbS 35.2%; HbF < 0.8%). The child was examined at ages 5, 11, and again at age 16 months. He is well, has maintained normal growth parameters without splenomegaly or any other SCD-related issues, is on penicillin prophylaxis but not on HU nor has he been transfused. At 11 months’ old, his Hb was 11.9 g/dL, HbS was 48.2%, and HbF was 50.7% (Table S1). His reticulocyte counts were between 1.8% and 2.5%; no other hemolytic markers were examined. Patient #2: This boy's neonatal hemoglobin screening revealed a Hb FS pattern. His mother is from the Dominican Republic and has sickle cell trait; his father is from Guatemala, and his hemoglobin electrophoresis showed HbA 88%, HbA2 2.9%, and HbF 9.1%. This child had a documented pain crisis at age 4 years after swimming in cold water, and another pain episode at age 6. He contracted parvovirus B19 infection and aplastic crisis at age 6 from which he recovered without a packed red blood cells (PRBC) transfusion. At age 8, he had salmonella bacteremia and osteomyelitis of the right femur and was hospitalized. He developed acute chest syndrome during hospitalization and required exchange transfusion. He has fully recovered and continues to do well. His Hb was 11.5 g/dL, HbS 61.9%, and HbF 35.7% (Table S1). He is not on any disease-modifying treatment. Patient #3: This girl suffered from failure to thrive and frequent pain in her extremities which required analgesics, starting from age 1 year. Her mother is of Spanish descent; her father of Portuguese ancestry. Their laboratory findings are shown in Table S2. At age 8, she had osteomyelitis of her left femur, was hospitalized, and treated with surgical intervention and antibiotics. No micro-organism was cultured. At age 9, she was diagnosed to have HbS/Sicilian (δβ)0-thalassemia deletion. She was started on HU at 20 mg/kg/day, followed by clinical improvement. At age 9½, her LD was 280 U/L; total bilirubin was 1.54 mg/dL. At age 11, she continues on HU and is doing well. Her Hb was 13.1 g/dL, HbS was 54.5%, and HbF was 39.3% (Table S1). At age 11½, her LD was 277 U/L; indirect bilirubin was 0.8 mg/dL. Her spleen was modestly enlarged. She has never been transfused. Results of β-globin gene nucleotide sequencing in these three children were consistent with either homozygosity or hemizygosity for HbS (data not shown). The presence of microcytosis without α-globin gene deletion and with elevated HbF suggested possible β-globin gene deletion. Multiplex ligation-probe amplification (MLPA) revealed a deletion involving δ- and β-globin genes, possibly Sicilian (δβ)0-thalassemia deletion (data not shown). A gap-polymerase chain reaction (PCR) test designed specifically for this deletion was done. The results confirmed that all three children were compound heterozygous for HbS and Sicilian (δβ)0-thalassemia deletion (Figure 1A,B). HbS haplotyping was done by genotyping five informative SNPs (Table S3). Patient #1 had the Arab-Indian HbS haplotype consistent with his being of Asian Indian descent; patient #2 had the Benin haplotype; patient #3 had the Bantu (CAR) haplotype. The Sicilian (δβ)0-thalassemia deletions found in these three unrelated children had identical haplotype, different from all major HbS haplotypes. The blood counts, hemoglobin analyses by capillary electrophoresis, genetic analyses of their β-globin genotypes, and F-cells determination by flow cytometric analysis were carried out in patient #3 and her parents (Table S2; Figure 1C). The father is heterozygous for the Sicilian (δβ)0-thalassaemia deletion and has heterocellular distribution of HbF, with F-cells accouting for 40.3%. The mother is heterozygous for HbS and has few F-cells accounting for 2.93%. Their daughter (patient #3) is compound heterozygous for HbS/Sicilian (δβ)0-thalassemia deletion. She has pancellular distribution of HbF, with F-cells accounting for 92.9%. Sicilian (δβ)0-thalassemia deletion is a deletion of 13.4 kb extending from IVS II of the δ-globin gene to a L1 element 3′ downstream of β-globin gene (Figure 1B).2, 3 Adult carriers of this deletion have microcytosis, elevated HbF (9.6 ± 3.5%), and decreased HbA2.3, 4 The fathers of all three patients have HbF of 10.6%, 9.1%, and 8.6%, respectively, consistent with their being heterozygous for the Sicilian (δβ)0-thalassemia deletion. All three patients had markedly elevated HbF. Patient #1 had 50.7% HbF at 11 months of age. He had the Arab-Indian HbS haplotype. The mean HbF level in HbSS patients with the Arab-Indian haplotype is around 18%, much higher than any of the four other major HbS haplotypes found in patients of African descent. Patients #2 and #3 had Benin and Bantu haplotypes, respectively. The mean HbF level in HbSS patients with these haplotypes is in the range between 5% and 7%. Patients with SCD and elevated HbF usually have mild disease. All three children reported here had HbF levels of 50.7%, 35.7%, and 39.3%, respectively. Patient #1 is well at age 16 months. Additional follow-up is needed to ascertain his disease severity. Patient #2 had history of rare pain episodes and developed salmonella bacteremia, osteomyelitis, and acute chest syndrome at age 8 years. Patient #3 had repeated pain episodes since age 1 and developed osteomyelitis at age 8. In patients #1 and #3, their reticuloctye counts, LD, and bilirubin levels were not elevated as in patients with brisk hemolysis. Patient #3 responded well to treatment with HU, with resolution of vaso-occlusive pain and improved growth and well-being. Her HbF levels prior to HU treatment were not known but likely elevated as in the other two children. After HU, her HbF levels were not significantly different from patient #2 (Table S1). The clinical beneficial effect of HU is thought to be primarily due to the up-regulation of HbF expression. However, HU can cause decreases in neutrophil and platelet counts, enhance nitric oxide production, downregulate endothelial adhesion molecules, and alter SCD erythrocyte membrane structure.5 It is conceivable that one or more of the above might have led to clinical improvement in patient #3. F-cell distribution was determined by flow cytometry in patient #3 and her parents. Her father is heterozygous for the Sicilian (δβ)0-thalassemia deletion and has heterocellular distribution of HbF in his peripheral blood erythrocytes (Figure 1C). In contrast, patient #3 has pancellular distribution of HbF (Figure 1C). Another girl with HbS/Sicilian (δβ)0-thalassemia deletion was reported in 2017 (Patient #4 in Table S1). At 1 year, her HbF was 41.2%. At 18 months of age, she remained well and was not on HU. This is the only case confirmed with DNA-based diagnosis reported in the literature. Cancio et al. reported nine children who were 4–17 years old with HbS/black (Aγδβ)0-thalassemia.6 Their mean Hb was 11.7 ± 1.5; HbF was 26.2 ± 4.3%. All of them suffered from SCD-related complications including acute chest syndrome and avascular necrosis. An 18-year-old African American young man with HbS/black HPFH 2 (Hb 13.9 g/dL; HbF 30.3%) developed massive splenic infarction.7 HbSE disease might be comparable to HbS/β+-thalassemia. Children and adolescents with HbSE disease are usually well. However, more than half of those aged 20 years and older have SCD-related complications.8 HbF is the most important genetic modifier to modulate SCD disease severity, based on its ability to inhibit deoxy-HbS polymerization. It is estimated that 30% or approximately 10 pg of HbF in each HbSS erythrocyte is optimal for the inhibition of deoxy-HbS polymerization. Pancellular distribution of HbF in HbSS erythrocytes is essential to achieve clinical benefit.9 Similar to other cases published in the literature, two of the 3 children reported here suffered from SCD complications in spite of markedly elevated HbF levels with pancellular distribution. These clinical observations further illustrate the complexity of SCD pathophysiology.10 It is conceivable that in patients #2 and #3, while their HbF distribution is pancellular based on flow cytometric analysis, yet HbF present in each erythrocyte is highly variable, some with a lot and others with little which might not be sufficient to negate deoxy-HbS polymerization leading to sickling and related complications. Reliable assay for HbF quantitation in individual erythrocyte is needed to validate this hypothesis. We thank Prof. Swee-Lay Thein for her insightful suggestions. All authors have no conflict of interest to declare. Banu Aygun, Adriana Bello, and Alexis A. Thompson cared for the children and provided clinical and hematology laboratory findings. Lance Davis, Yanan Sun, and Hong-yuan Luo undertook diagnostics. Shuaiying Cui and David H. K. Chui analyzed the data and wrote the initial draft of the manuscript. All authors edited the manuscript and approved the final version. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Table S1 Hematology laboratory results. Table S2 Laboratory results of patient #3 and her parents. Table S3 HbS haplotype. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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