Abstract
Hemoglobin S/Black (Aγδβ)0-thalassemia is a rare sickle cell disease (SCD) variant. Based on limited descriptions in the literature, the disease is reported as a mild microcytic anemia with an uncomplicated course. We report the clinical and laboratory data of nine patients whose diagnoses were confirmed by DNA-based techniques. Despite having mild anemia and high fetal hemoglobin level post-infancy, these patients developed many of the classic complications of SCD, including vaso-occlusive crisis, acute chest syndrome, avascular necrosis, and cholelithiasis. Based on these findings, we recommend that patients with this rare disorder receive specialized hematology care according to SCD guidelines.
Keywords: Sickle cell disease, sickle cell anemia, thalassemia, sickle gamma delta beta thalassemia, Gγ(Aγδβ)0-thalassemia
INTRODUCTION
A-gamma delta beta thalassemia is a rare form of thalassemia resulting from large deletions, including the Aγ, δ and β globin genes. There are at least 11 different forms reported,[1] one of which, the Black(Aγδβ)0-thalassemia, may occur in association with sickle hemoglobin (HbS). Laboratory and clinical characterizations of Black(Aγδβ)0-thalassemia (homozygous and compound heterozygous) have been reported, yet little information exists for the compound heterozygotes with HbS, a rare variant of sickle cell disease (SCD) that is considered to have a mild clinical course as previously reported in only 7 individuals.[2] HbS/Black(Aγδβ)0-thalassemia occurs primarily in persons of African ancestry and is associated with high fetal hemoglobin (HbF) level, mild anemia, and slight microcytosis with hypochromia. Herein we describe the hematological and clinical course of nine children with HbS/Black(Aγδβ)0-thalassemia who, contrary to the existing literature, developed many of the complications of SCD.
CASE DESCRIPTIONS
With institutional review board approval, a search of clinical databases of three SCD programs between 2000 and 2014 identified nine patients with HbS/Black(Aγδβ)0-thalassemia. Each patient underwent hemoglobin analysis by Bio-Rad Variant II high performance liquid chromatography and confirmation by gap-PCR test and nucleotide sequencing in the Hemoglobin Diagnostic Reference Laboratory (Boston Medical Center). Six of the nine patients (P1, P4, P5, P6, P7, P8) were identified through newborn screening (HbFS and absent HbA, Table 1). The remaining patients were referred in their second decade of life, following identification through sickle cell trait screening (P2 and P3), and referral by a rheumatologist (P9) who noted hip pain and mild microcytic anemia not attributed to a rheumatologic disorder. These nine patients had in common persistently elevated HbF levels (>20% after age 1), partially compensated hemolytic anemia, and all underwent comprehensive hemoglobinopathy testing to investigate the etiology of HbF elevation. Molecular testing identified a deletion of 35.8 kb with the 5’ breakpoint in IVSII of the Aγ globin gene and the 3’ breakpoint at the L1 repeat downstream of the β globin gene (Supplemental Figure S1).
Table 1.
Demographics and Laboratory Parameters at Diagnosis and Last Follow-Up.
| Patient | Gender | Age(years) | Hb(g/dL) | MCV(fL) | ARC(x103/) | Total bilirubin (mg/dL) |
Hb identification(%) | |
|---|---|---|---|---|---|---|---|---|
| 1 | M | 0.08 | 12.1 | 71 | 0.055 | n/a | 88.9 | 6.3 |
| 13.08 | 11.3 | 73.8 | 0.127 | n/a | 25.2 | n/a | ||
| 2 | M | 17.5 | 13.8 | 77.2 | 0.106 | 1.6 | 21 | 75.8 |
| 23.9 | 12.3 | 72.5 | n/a | n/a | 24.7 | 66.5 | ||
| 3 | M | 17.5 | 13.6 | 78.2 | 0.091 | 2 | 21.4 | 75.4 |
| 22.9 | 14.7 | 74.8 | n/a | n/a | 21.8 | n/a | ||
| 4 | M | 0.13 | 10.6 | 84.3 | 0.113 | n/a | 74.7 | 13.3 |
| 6.3 | 11.6 | 72.9 | 0.144 | n/a | 26.3 | 64.3 | ||
| 5 | F | 0.5 | 11.4 | 72.7 | 0.109 | 0.2 | 34 | 47.6 |
| 4.0 | 10.7 | 66.8 | n/a | n/a | 26.5 | 64.8 | ||
| 6 | F | 1 | 13.3 | 71 | 0.204 | 0.6 | 25.9 | 71.1 |
| 11 | 12.3 | 70 | 0.162 | n/a | 24.2 | 74.8 | ||
| 7 | M | 0.12 | 10.2 | 86 | 0.365 | 0.4 | n/a | n/a |
| 4.8 | 11.7 | 70 | 0.072 | 1.6 | 34 | 63.5 | ||
| 8 | M | 0.5 | 10.8 | 64 | 0.146 | n/a | 32 | 65.7 |
| 7 | 8.3 | 65 | 0.188 | n/a | 32.8 | 64.7 | ||
| 9 | M | 12 | 11.9 | 66 | 0.084 | 1.5 | 24 | 73 |
| 17 | 14.2 | 81 | n/a | n/a | n/a | n/a | ||
Notes: M: Male; F: Female; Hb: hemoglobin; MCV: mean corpuscular volume; ARC: absolute reticulocyte count; n/a: not available. Hb identification was performed using high performance liquid chromatography. HbA was zero in all patients.
HbS/Black(Aγδβ)0-thalassemia children had mild microcytic anemia, reticulocytosis, and hyperbilirubinemia (Table 1). Peripheral blood smear disclosed anisocytosis, poikilocytosis, microcytosis, target cells, and rare sickled forms (Supplemental Figure S2). Prophylactic penicillin was provided to all diagnosed in the newborn period and most continued this until age 5.
P1 had dactylitis at age 7 months. No other vaso-occlusive crisis (VOC) events were recorded at the time of last follow-up, at age 13. Analysis by flow cytometry showed 94.8% HbF positive cells (Supplemental Figure S3).
P2 and P3 were prematurely-born twins who were referred at age 17 years, after sickle cell screening at a health fair. They had multiple hospital visits for management of severe lower extremity pain in the absence of trauma or other inciting events, both before and after diagnosis. These episodes required intravenous hydration and analgesia. P3 underwent cholecystectomy for symptomatic cholelithiasis at age 19.
Patient 4 had dactylitis at age 2. He required hospitalization twice between the ages of 2 and 3 for acute chest syndrome (ACS; fever, pain, cough, and a new pulmonary infiltrate), without requirement for supplemental oxygen. Therapy with hydroxyurea was initiated with subsequent improvement of VOC symptoms. Qualitative HbF analysis by flow cytometry showed 91.7% HbF positive cells (Supplemental Figure S3).
P5 had no sickle related complications at last follow-up at age 4.
P6 had mild splenomegaly, multiple episodes of VOC by age 11, and recurrent episodes of abdominal pain (without documentation of cholelithiasis) concurrent with pain in extremities.
P7 had frequent complains of abdominal pain, which after extensive gastrointestinal evaluation, were attributed to constipation. No VOC events were recorded by his last follow-up at age 4.
P8 was admitted for fever and first episode of leg pain at age 7; magnetic resonance imaging (MRI) was suggestive of osteomyelitis. His symptoms improved within 4 days of antibiotics, recurred upon premature discontinuation, but resolved when antimicrobials were resumed.
By age 9, P9 had splenomegaly and multiple admissions for severe recurrent pain in the upper and lower extremities and back, consistent with VOC. MRI revealed multifocal avascular necrosis affecting both hips and right shoulder. Hydroxyurea was initiated resulting in fewer VOC episodes.
During the period of this analysis (60.7 patient-years), SCD-related complications occurred in seven out of nine patients. These complications included VOC, dactylitis, cholelithiasis, osteonecrosis, ACS, and osteomyelitis. There were no instances of splenic sequestration, stroke or sepsis. No erythrocyte transfusions were required for SCD-related complications. Additionally, normal transcranial Doppler ultrasound velocities were noted in three patients (P1, P4, P5), and three had obstructive sleep apnea (P5, P8, P9). Laboratory values were plotted as a function of age; HbF declined after birth, but overall remained at approximately 20%, mean corpuscular volume (MCV) remained stable, and Hb concentration increased slightly with age (primarily driven by teenagers P2 and P3 who entered the cohort at age 17) (Figure 1).
Figure 1. Hematologic parameters of patients with Hemoglobin S/Black (Aγδβ)0-thalassemia by age.
Hemoglobin (Hb) remained stable and above 9 g/dL, mean corpuscular volume (MCV) was low or low normal for age, and fetal hemoglobin (HbF) declined but remains above 20% with aging. Age was grouped in 2-year intervals.
DISCUSSION
The compound heterozygous HbS/Black(Aγδβ)0-thalassemia produces a rare SCD variant that, until this date, had only been described in 7 cases worldwide.[2] Here we describe the clinical course of nine patients from birth to age 24 with HbS/Black(Aγδβ)0-thalassemia whose molecular characterization shows distinct beta globin locus breakpoints from the benign compound heterozygous HbS and hereditary persistence of fetal hemoglobin (HbS/HPFH), but also exhibits high HbF levels (Supplemental Figure S1). Their course was not always as benign as reported in the literature or generally assumed, with SCD-related complications occurring from infancy into young adulthood.
HbS/Black(Aγδβ)0-thalassemia was described in 1985, when restriction enzyme analysis was used to study members of 10 families with conditions considered to be Gγ(δβ)0 -thalassemia or Gγ(δβ)0-HPFH. Seven heterozygotes with HbS/Black(Aγδβ)0-thalassemia were identified; these patients were reported to have a mild variant of SCD.[2] Their average HbF was 19.2% but the pattern of HbF distribution was not reported.
Our data are limited by the small sample size and limited follow-up, but it is the largest reported to date of this condition. Due to the rarity of the condition and limited clinical data available, there is no information regarding the type of routine care patients with HbS/Black(Aγδβ)0-thalassemia should receive. In addition to having mild microcytic anemia and hemolysis, patients with HbS/Black(Aγδβ)0-thalassemia are at risk for SCD-related complications, and their clinical course seems to approximate that of individuals with HbSβ+ thalassemia, a relatively milder SCD phenotype. This report highlights the importance of definitive diagnosis with DNA-based testing when HbF level is elevated, but laboratory tests do not support the diagnosis of other relatively benign disorders such as HbS/HPFH (e.g. moderate hemolysis, supplemental Table S1).[3]
It is unclear why patients with HbF >20% should have high frequency of SCD-related complications, but symptomatic disease has been described in similar SCD variants with elevated HbF.[4,5] Alleviation of SCD symptoms in patients with the benign variant with HbF>20%, HbS/HPFH, is associated with > 99% distribution of HbF-expressing cells (F-cells).[6] The positive F-cell fraction measured by flow cytometry in two of our symptomatic patients was 92–95%. These results were interpreted as pancellular, although a discrete population of HbF negative cells was detected in both patients (Supplemental Figure S3). Therefore, it is possible that the small percentage of HbF-negative cells in the HbS/Black(Aγδβ)0-thalassemia reflects heterogeneity in HbF expression, which could potentially explain the phenotype differences between HbS/Black(Aγδβ)0-thalassemia and HbS/HPFH populations.
Our data suggest that children with HbS/Black(Aγδβ)0-thalassemia may have SCD-complications and therefore should be monitored by SCD specialists in order to minimize morbidity and potential mortality. Recommendations applicable to patients with SCD, e.g., routine laboratory testing, stroke screening, hydroxyurea therapy, penicillin prophylaxis, and immunizations against encapsulated organisms, should be extended to patients with HbS/Black(Aγδβ)0-thalassemia.
Supplementary Material
Supplemental Figure S1. Black (Aγδβ)0-thalassemia deletion, Black HPFH-1 and HPFH-2 deletions. The top row depicts the globin and olfactory receptor genes. Red denotes active genes; blue denotes pseudo-genes. The numbers on the second row represent positions on chromosome 11p (hg19). The third row displays functional motifs. The Black (Aγδβ)0-thalassemia deletion’s 5’ breakpoint is within the 2nd intron of the Aγ-globin gene. The deletion extends for 35.8 kb downstream and is distinct from that of Black HPFH-1 and 2.
Supplemental Figure S2. Peripheral Blood smear of patient with Hemoglobin S/Black (Aγδβ)0-thalassemia. Anisocytosis is noticed, in addition to target cells (black arrow), sickle cell (red arrow), and hypochromia (blue arrow).
Supplemental Figure S3. Hemoglobin F Red Cell Distribution. Flow cytometry analysis shows HbF distribution for two patients with Hemoglobin S/Black(Aγδβ)0-thalassemia. Both patients had a small proportion of negative HbF-staining cells despite the high HbF% values at the time of analysis (Patient 1 had HbF 25.2% and Patient 4 HbF 26.3% as measured by HPLC). Flow cytometry analysis of HbF distribution was performed by Mayo Clinic Laboratories.
ACKNOWLEDGEMENTS
This work was supported in part by the American Lebanese Syrian Charities (ALSAC). The authors would like to thank Jennifer Oliveira, MD and the Hematopathology laboratory at Mayo Clinic for supplying flow cytometry figures, Mitchell Weiss, MD, PhD for helpful discussions during preparation of this manuscript, Matthew Smeltzer, MS, PhD for support with data interpretation, and Jason Hodges, PhD for preparing some of the figures. We also thank Joseph Olechnowicz for editorial assistance. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748.
Abbreviations
- SCD
sickle cell disease
- HbS
sickle hemoglobin
- HbF
fetal hemoglobin
- VOC
vaso-occlusive crisis
- OSA
obstructive sleep apnea
- MCV
mean corpuscular volume
Footnotes
Conflicts of interest: The authors have no conflicts of interest to declare.
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Associated Data
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Supplementary Materials
Supplemental Figure S1. Black (Aγδβ)0-thalassemia deletion, Black HPFH-1 and HPFH-2 deletions. The top row depicts the globin and olfactory receptor genes. Red denotes active genes; blue denotes pseudo-genes. The numbers on the second row represent positions on chromosome 11p (hg19). The third row displays functional motifs. The Black (Aγδβ)0-thalassemia deletion’s 5’ breakpoint is within the 2nd intron of the Aγ-globin gene. The deletion extends for 35.8 kb downstream and is distinct from that of Black HPFH-1 and 2.
Supplemental Figure S2. Peripheral Blood smear of patient with Hemoglobin S/Black (Aγδβ)0-thalassemia. Anisocytosis is noticed, in addition to target cells (black arrow), sickle cell (red arrow), and hypochromia (blue arrow).
Supplemental Figure S3. Hemoglobin F Red Cell Distribution. Flow cytometry analysis shows HbF distribution for two patients with Hemoglobin S/Black(Aγδβ)0-thalassemia. Both patients had a small proportion of negative HbF-staining cells despite the high HbF% values at the time of analysis (Patient 1 had HbF 25.2% and Patient 4 HbF 26.3% as measured by HPLC). Flow cytometry analysis of HbF distribution was performed by Mayo Clinic Laboratories.