Association of alpha‐thalassemia and Glucose‐6‐Phosphate Dehydrogenase deficiency with transcranial Doppler ultrasonography in Nigerian children with sickle cell anemia

Abstract Background Stroke is a devastating complication of sickle cell anemia (SCA) and can be predicted through abnormally high cerebral blood flow velocity using transcranial Doppler Ultrasonography (TCD). The evidence on the role of alpha‐thalassemia and glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in the development of stroke in children with SCA is conflicting. Thus, this study investigated the association of alpha‐thalassemia and G6PD(A−) variant with abnormal TCD velocities among Nigerian children with SCA. Methods One hundred and forty‐one children with SCA were recruited: 72 children presented with normal TCD (defined as the time‐averaged mean of the maximum velocity: < 170 cm/s) and 69 children with abnormal TCD (TAMMV ≥ 200 cm/s). Alpha‐thalassemia (the α‐3.7 globin gene deletion) was determined by multiplex gap‐PCR, while G6PD polymorphisms (202G > A and 376A > G) were genotyped using restriction fragment length polymorphism—polymerase chain reaction. Results The frequency of α‐thalassemia trait in the children with normal TCD was higher than those with abnormal TCD: 38/72 (52.8%) [α‐/ α α: 41.7%, α ‐/ α ‐: 11.1%] versus 21/69 (30.4%) [α‐/ α α: 27.5%, α ‐/ α ‐: 2.9%], and the odds of abnormal TCD were reduced in the presence of the α‐thalassemia trait [Odds Ratio: 0.39, 95% confidence interval: 0.20–0.78, p = 0.007]. However, the frequencies of G6PDA− variant in children with abnormal and normal TCD were similar (11.6% vs. 15.3%, p = 0.522). Conclusion Our study reveals the protective role of α‐thalassemia against the risk of abnormal TCD in Nigerian children with SCA.


| INTRODUC TI ON
Stroke is a severe complication of sickle cell anemia which may result in a permanent neurocognitive impairment and physical dysfunction. 1 Although the risk of stroke persists throughout the entire life of persons living with SCA, it is more likely in the first decade of life. 2,3 The incidence of ischemic stroke is 1.02% per year between 2 and 5 years old while hemorrhagic stroke is predominant between 20-29 years of age; the latter is believed to be caused by stenosis of major intracranial vessels leading to moya-moya or by the formation of aneurysms in the vessels of the circle of Willis. 4,5 An ischemic stroke usually results from occlusion of large cerebral arteries with an interplay of several mechanisms involved in hemolysis, hypoxia, anemia, abnormal red blood cell rheology, inflammation, endothelial dysfunction, and genetic factors. 6 Previous studies have also identified prior transient ischemic attack, anemia, a past history or increased frequency of acute chest syndrome, and elevated systolic blood pressure as independent predictors of ischemic stroke, while anemia and high white blood cell count were also found to be independently associated with hemorrhagic stroke. 1,4 Significant progress has been made in the prevention of first stroke hydroxyurea and blood transfusion. 7 Unlike chronic blood transfusion which is fraught with many challenges in low resource countries, hydroxyurea therapy has been shown to be effective for primary stroke prevention in Nigerian children with SCA. 8,9 Alpha-thalassemia is characterized by deletional loss of one or both alpha-globin genes. α-3.7 kb thalassemia deletion is the most common alpha-thalassemia deletion in sub-Sahara African populations and is a well-known genetic modifier of SCA. Varied prevalence of α-thalassemia deletion (α 3.7 ) has been reported from different populations among persons with SCA: 30% in Brazil, 37% in Cameroon, 41-43% in Nigeria, 48% in France, and 56% in Tanzania. [10][11][12][13][14][15] Co-inheritance of α 3.7 -thalassemia with SCA is associated with increased risk of avascular necrosis and vaso-occlusive crises while it improves survival, reduces rate of leg ulcers, the need for blood transfusion, and stroke risk. 11,16,17 Some studies suggest that the protective effect of alpha-thalassemia against stroke and abnormal TCD velocity may be related to the reduced hemolytic rate and improved rheological properties of red blood cells. 14,18 Glucose-6-phosphate dehydrogenase (G6PD) is an important source of Nicotinamide adenine dinucleotide phosphate (NADPH) in pentose phosphate pathway and is present in both erythrocytes and endothelial cells. 19,20 G6PD deficiency participates in increased generation of reactive oxygen species and decreased nitric oxide (NO) bioavailability and its impact on SCA may worsen vascular dysfunction and contribute significantly to cerebral vasculopathy. 20,21 G6PD deficiency is common in sub-Saharan Africa and the predominant alleles: 202A and 376G as G6PDA − are responsible only for approximately 12% of enzyme activity compared to the wild-type 202G and 376A G6PD B allele. 22 It has been suggested that G6PD deficiency worsens anemia and is linked to the increased need for blood transfusion, playing a significant role in the pathophysiology of cerebral vasculopathy. 23 Findings on the association of alpha-thalassemia deletion and G6PD deficiency with abnormal TCD velocity in children with sickle cell anemia from different populations are conflicting, suggestive of geographical and genetic modifiers. 13,14,[24][25][26] Despite the high burden of stroke risk and stroke in Nigerian

| Study design
In this study, we enrolled 141 children with SCA (2-15 years old) attending the Sickle Cell Foundation Nigeria, Lagos for TCD screening as part of their routine care over a 6 month period. The sickle cell anemia status of each enrollee was confirmed by alkaline electrophoresis to determine the hemoglobin SS (Hb SS) phenotype and solubility test to ascertain sickling characteristics of Hb SS in a deoxy-state following the standard quality control procedures.
Other inclusion criteria included being in a stable condition and not more than 16 years old at enrollment. Children who were not in steady-state at presentation, aged >16 years old, had hemoglobin C phenotype, on hydroxyurea, received blood transfusion in the previous 4 months, or had clinical history of stroke were excluded from this study. Also excluded from the study were children with intermediate (conditional) stroke risk characterized by TAMMV of 170-199 cm/s.

| TCD screening
Non-imaging TCD was measured for all the children according to the Stroke prevention trial (STOP) guidelines as previously described 27 and the time-averaged mean of maximum velocities (TAMMV) were recorded. Children with TAMMV < 170 cm/s were defined as having normal TCD and represented a low stroke risk group, while children with TAMMV > 200 cm/s were defined as having abnormal TCD and represented high stroke risk group. 27

| Pulse oximetry and laboratory analyses
Pulse oximeter was used to measure hemoglobin oxygen saturation (SpO 2 ) of the children after TCD scan. Full blood counts were determined using an automated hematology analyzer (Mindray, BC-2800, China). Fetal hemoglobin was evaluated by high-performance liquid chromatography (D-10 Hemoglobin testing system, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Lactate dehydrogenase (LDH) activity was assayed spectrophotometrically at 340 nm with a Cobas kit (Roche/Hitachi analyzer, Roche Diagnostics GmbH).
Percentage reticulocyte count was determined microscopically using blood films pre-stained with new methylene blue.

| Genetic analyses
A 3.7 kb alpha-globin gene deletion was genotyped by multiplex gap-PCR 28 while glucose-6-phosphate dehydrogenase polymorphisms (202G > A and 376A > G) were genotyped using restriction fragment length polymorphism (RFLP-PCR). 29 Children with one (heterozygous) or two (homozygous) α-globin gene deletions were regarded as having alpha-thalassemia trait while they were classified as G6PD deficient (G6PDA − ) or normal (G6PDA or G6PD B) based on the presence or absence of 202A/376G genotype.

| Ethical considerations
Ethical approval was obtained from the Lagos University Teaching hospital health research and ethics committee (ADM/DCST/ HREC/843). Written informed consent from parents/guardians and assent of the children who were 7 years old and above were obtained before enrolment.

| Statistical analysis
Data obtained were summarized in percentages and mean ± standard deviation. The frequencies of the wild (G6PD202A/376G) and deficient (G6PD202G/376A) G6PD, as well as the alpha-thalassemia trait observed, characterized by heterozygous and homozygous deletions (α α /α andα /α) versus normal α-globin condition (α α / α α), were determined by counting the corresponding DNA bands on agarose gels. Categorical variables between the abnormal TCD and normal TCD groups were compared by chi-square (χ 2 ) and Fisher's exact tests, while differences between continuous variables were evaluated using Student's t test. Both the odds ratio (OR) and 95% confidence interval (95%CI) were also calculated. Multivariable logistic regression was also performed to determine independent predictor(s) of abnormal TCD. All statistical analyses were carried out using SPSS version 25 for Windows. Statistical outcomes with p-value <0.05 were considered significant.

| RE SULTS
The mean age of children with normal and abnormal TCD was 8.4 ± 3.6 and 7.0 ± 3.5 years old (range: 2-15 years), respectively.
Percentage SpO 2 was significantly lower in children with abnormal TCD compared to normal TCD (p = 0.007) while WBC, MCV, MCH, and LDH were significantly higher in the abnormal TCD group than the normal TCD group (p < 0.01); the reticulocyte count, however,

TA B L E 1 Clinical and laboratory parameters of children with SCA
was not significant (p = 0.068) (

| DISCUSS ION
In this study, we found significantly lower hemoglobin oxygen satu-  cell anemia was reported. 14,26 In both studies, the protective role of alpha-thalassemia trait was linked to its association with reduced hemolysis among their sickle cell cohorts. Another mechanism that has been described is the association of alpha-thalassemia trait with reduced exposure of phosphatidylserine on the surface of erythrocyte membrane, thereby reducing red cell adhesion to the endothelium and reduced vascular injury. 18,32 In contrast to our study, abnormal TCD measured according to STOP protocol did not show association with alpha-thalassemia trait in the Tanzanian children with sickle cell anemia but confirmed protective effect of alpha-thalassemia against abnormally low cerebral blood flow velocity. 15 It is likely that the higher frequency of alpha-thalassemia trait in the Tanzanian cohort, which is linked with the Bantu β-globin haplotype accounts for the lower prevalence of abnormal TCD. 15,33 Meanwhile, higher prevalence of 10-16% abnormal TCD has been reported in other studies. 3,14 G6PD deficiency has been linked to increased accumulation of reactive oxygen species, depletion of glutathione stores, and enhanced oxidant stress, which in turn leads to decreased bioavailability of nitric oxide and vascular dysfunction. 19 Previous studies had found no evidence that G6PD deficiency influences the severity of hemolysis or increases the incidence of acute anemic events in persons with SCA. 14,34 However, others have shown that the risk of an abnormal TCD was associated with increased G6PD deficiency. 14, 26 We did not find an association between G6PDA − and abnormal TCD in our cohort. Our finding is in agreement with the report of Belisario et al 25 who also found that G6PD deficiency did not influence stroke risk or occurrence of ischemic stroke among Brazillian children with SCA. One major difference between our studies and the French cohorts is that G6PD deficiency was determined by G6PD enzyme activity and not by G6PDA-molecular deficiency presented in this study. 14,26 Also, most studies did not find an association of abnormal TCD/stroke with G6PD deficiency using G6PD enzyme activity or G6PDA − variant. 24,25,35,36 Our study has some limitations. First, our sample size is small, and it is possible that a larger study would provide better power to detect associations. Secondly, the study was a single-center design which may limit the generalization of the findings, but the Sickle Cell Foundation Lagos offers clinical services to patients referred from primary, secondary, and tertiary clinics in Lagos, Nigeria. Thirdly, only single and double 3.7 kb α-globin gene deletions were investigated in this study. Besides, we assessed only G6PD deficiency (A − ) and did not examine other forms of G6PD polymorphisms that may be present in our population. Large cohorts will be needed in future studies to validate the relationship between TCD and alphathalassemia trait/G6PD deficiency.

ACK N OWLED G EM ENTS
We are grateful to the children who volunteered to be part of this study. We are also grateful to the TCD technicians at the Sickle Cell Foundation Nigeria who performed the transcranial Doppler screening.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.