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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Br J Haematol. 2010 May 9;150(2):218–225. doi: 10.1111/j.1365-2141.2010.08215.x

Association of G6PD202A,376G with lower haemoglobin concentration but not increased haemolysis in patients with sickle cell anaemia

Mehdi Nouraie 1, Noel S Reading 2, Andrew Campbell 3, Caterina P Minniti 4, Sohail R Rana 1, Lori Luchtman-Jones 5, Gregory J Kato 4, Mark Gladwin 6, Oswaldo L Castro 1, Josef T Prchal 7, Victor R Gordeuk 1
PMCID: PMC2906678  NIHMSID: NIHMS212496  PMID: 20507315

Abstract

The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD202A and G6PD376G alleles and α-thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD202A,376G (G6PD A-) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide.

The prevalence of G6PD202A,376G was 13.6% in males and 3.3% in females with an overall prevalence of 8.7%. G6PD202A,376G was associated with a 10 g/l decrease in haemoglobin concentration (P=0.008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate-aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P>0.09). Similar results were found within a sub-group of children who were not receiving hydroxycarbamide. By comparison, single and double α-globin deletions were associated with progressively higher haemoglobin concentrations (P=0.005 for trend), progressively lower values for haemolytic component (P=0.007), and increased severe pain episodes (P<0.001).

Conclusions

In conclusion, G6PD202A,376G may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.

Keywords: sickle cell anaemia, G6PD, haemolysis, alpha-thalassaemia, haemoglobin concentration

Introduction

Individuals with sickle cell anaemia, i.e., homozygotes for the haemoglobin S mutation, have variability in the severity of anaemia and degree of haemolysis at steady state (Steinberg and Hebbel 1983), and the reasons for this variability are not fully known (Christakis, et al 1990). α-Thalassaemia due to single or double HBA1/2 deletions (Chaar, et al 2006, Embury, et al 1982, Higgs, et al 1982) and higher haemoglobin F (Hb F) percentages (Franco, et al 2006, Maude, et al 1987, Serjeant 1975) are associated with higher haemoglobin concentrations and less haemolysis in this setting.

The glucose-6-phosphate dehydrogenase (G6PD) gene (GSPD) is located on the X chromosome. The normal allele is termed G6PD B. Mild G6PD deficiency (G6PD A-) has a prevalence of about 10% in predominantly African-American United States sickle cell disease patients based on reduced enzyme activity (Steinberg, et al 2003). The G6PD A- phenotype is usually caused by hemizygosity for both the G6PD202A and G6PD376G alleles in males and homozygosity for both of these alleles in females (Hirono and Beutler 1988) and is referred to G6PD202A,376G in this report. Hemizygosity for G6PD376G but not G6PD202A is termed G6PD A and is usually associated with normal enzymatic activity. G6PD202A,376G has a prevalence of about 12–13% in males and 2–3% in females in sub-Saharan Africa (Guindo, et al 2007, Johnson, et al 2009) and accounts for most of the G6PD deficiency in the population as determined by enzymatic assay (Johnson, et al 2009). Other genotypes, particularly inheritance of both the G6PD376G and G6PD968C alleles (G6PD376G,968C), may contribute to the G6PD A- phenotype and even predominate over G6PD202A,376G, especially in certain parts of West Africa (Clark, et al 2009, De Araujo, et al 2006).

G6PD A- may be associated with episodic haemolysis in individuals of African descent and has a frequency among sickle cell anaemia patients that is similar to the general background population (Bienzle, et al 1975, Las Heras Manso, et al 2008, Segeja, et al 2008). Little influence of G6PD A- on the degree of anaemia and of haemolysis was reported in several series of sickle cell anaemia patients (Bernaudin, et al 2008, Gibbs, et al 1980, Steinberg, et al 1988). Here we examined the effect of G6PD202A,376G, as determined by molecular genetic testing, on the haemoglobin concentration and degree of haemolysis in a new cohort of children and adolescents with haemoglobin SS (Hb SS) at steady state, those enrolled in the Pulmonary Hypertension and the Hypoxic Response in sickle cell disease (PUSH) Study (Minniti, et al 2009).

Material and Methods

Clinical study of participants

Individuals 3 to 20 years of age with sickle cell anaemia (Hb SS) were recruited at three tertiary medical centres: Children’s National Medical Center and Howard University Hospital in Washington, DC, and University of Michigan in Ann Arbor, Michigan. Children were recruited as outpatients when they presented for routine, ongoing care. To ensure that patients were enrolled at steady state, at least three weeks had to have elapsed since hospitalization, emergency department visit, or clinic visit for acute chest syndrome, pain crisis, infection or other sickle cell disease-related complication. Written informed consent was obtained for all participants. Parents or legal guardians provided consent for children aged 3 to 17 years. Participants aged 18 to 20 years provided consent for themselves. Participants aged 7 to 17 years provided additional written informed assent. Participants receiving regular blood transfusions or hydroxycarbamide therapy were not excluded. Detailed descriptions of experimental methods were published recently (Gordeuk, et al 2009, Minniti, et al 2009).

Molecular genetic testing

G6PD: G6PD genotyping was conducted on a LightCycler480 (LC480) Real-Time PCR System (Roche Applied Science, Indianapolis, IN) using TaqMan custom probes (Applied Biosystems, Foster City, CA). In this report, G6PD202A,376G indicates boys who are hemizygous and girls who are homozygous for both the G6PD202A and the G6PD376G alleles (Table 1), and this genotype is strongly associated with glucose-6-phosphate dehydrogenase deficiency (Hirono and Beutler 1988). HBB: Haemoglobin S genotyping was conducted at ARUP Laboratories (Salt Lake City, UT) using loci-spanning probe – PCR as described (Pont-Kingdon, et al 2007). HBA1/2: α-thalassaemia genotyping was performed as described (Tan, et al 2001).

Table 1.

Phenotypic characteristics of different G6PD202,376 genotypes

Allele Amino Acids Common designation Enzymatic activity
202G,376A 68 Val, 126 Asn G6PD B Normal
202G,376G 68 Val, 126 Asp G6PD A Electrophoretic variant with normal activity
202A,376G 68 Met, 126 Asp G6PD A- Deficient variant
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Echocardiography

Transthoracic echocardiography was performed using the Philips Sono 5500/7500 or iE33, Acuson Sequoia, or General Electric VIVID 7 or VIVID I instruments. Systolic pulmonary artery pressure was estimated through measurement of tricuspid regurgitation velocity. Cardiac images were obtained, measurements performed, and studies interpreted centrally according to guidelines of the American Society of Echocardiography (Lai, et al 2006).

Statistical analysis

Principal component analysis of age-adjusted values for lactate dehydrogenase (LDH), asparate aminotransferase (AST), reticulocyte count and total bilirubin was used to develop a haemolytic component that predicted 63% of variability among these factors and had a mean of 0 and standard deviation (SD) of 1.52. The arbitrary units of this variable were converted to positive SD units with a mean of 3.0 and SD of 1.00. Principal component analysis was used when the objective of the study was to investigate the role of an underlying mechanism (Genser, et al 2007), in this case the degree of haemolysis. The development of such a component resolved the problem of dealing with correlated predictors in multivariate analyses. Continuous and categorical variables were examined according to different genotypes by linear test for trend, student’s t test or Chi-square. Multiple linear regression models were developed to identify the independent predictors of haemoglobin concentration and haemolytic component. These models were checked for model assumptions and outliers. . An arbitrary level of 5% statistical significance was assumed to be significant. Statistical analyses were performed with STATA 10.0 (StataCorp, College Station, TX).

Results

Demographics and genotypes

Two hundred and sixty-one Hb SS patients were studied for G6PD202A,376G and for HBA1/2 deletions. The median age was 12 years, 47% were females, and 98% were African Americans. The allele frequencies for G6PD376G (35.2%), G6PD202A (19.2%) and HBA1/2 (α-thalassaemia; 17.0%) were similar to the frequencies in 57 controls from the background population (data not shown). Seventy-nine (30%) of the Hb SS patients had α thalassaemia, 72 with single HBA1/2 deletion and seven with double HBA1/2 deletions.

Haemoglobin concentration and haemolytic component according to G6PD alleles

G6PD genotype and phenotype correlations are summarized in Table 1. Nineteen (13.8%) of the 138 male Hb SS patients were hemizygous for both mutant alleles, i.e. had G6PD202A,376G (G6PD A). Twenty-seven (19.6%) were hemizygous for G6PD376G but not G6PD202A, i.e. had G6PD202G,376G (G6PD A). Ninety-two (66.7%) had neither the G6PD376G nor the G6PD202A mutations, i.e. had G6PD202G,376A (G6PD B). Table 2 shows that hemizygotes for both G6PD376G and G6PD202A had lower haemoglobin concentration but not higher haemolytic component than the G6PD B normal (G6PD202G,376A) participants. In contrast, the males with G6PD A (hemizygotes for G6PD376G but not G6PD202A) did not have either significantly lower haemoglobin concentration or higher haemolytic component compared to the G6PD B group. In further analyses, the males with G6PD202A,376G were compared to the combined group of males with the other two genotypes.

Table 2.

Haemoglobin concentration and haemolytic component by different G6PD202,376 genotypes in males. Results are given asmean (standard error).

G6PD202G,376A (G6PD B) N=92 G6PD202G,376G (G6PD A) N=27 G6PD202A,376G (G6PD A-) N=19
Haemoglobin concentration (g/l), unadjusted 87 (1.5)1 84 (2.5) 79 (2.9)2
P* 0.3 0.022
Haemoglobin concentration (g/l), adjusted** 88 (1.4)1 84 (2.4) 77 (3.0)2
P* 0.1 0.001
Haemolytic component (relative unit), unadjusted 3.1 (0.1)3 3.3 (0.2)4 3.3 (0.2)5
P* 0.5 0.4
Haemolytic component (relative unit), adjusted*** 3.1 (0.1)3 3.3 (0.2)4 3.4 (0.3)5
P* 0.5 0.3
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*

Comparison with the G6PD202G,376A (G6PD B) group.

**

Adjusted for recent blood transfusion and haemolytic component.

***

Adjusted for hydroxycarbamide treatment and α-thalassaemia.

N: the number with the genotype in the data set.

1

N=86

2

N=18

3

N=80

4

N=25

5

N=17

Four (3.3%) of the 123 female Hb SS patients were homozygotes for both the G6PD376G and G6PD202A alleles, i.e. had G6PD202A,376G/202A,376G (G6PD A-). Twelve (9.8%) had G6PD376G together with G6PD202A on one allele but not the other, i.e. had G6PD202A,376G/202G,376A or G6PD202A,376G/202G,376G. Fifty-six (45.5%) had G6PD376G on at least one allele but not in combination with G6PD202A, i.e. had G6PD202G,376A/202G,376G or G6PD202G,376G/202G,376G. Fifty-one (41.5%) had G6PD202G,376A/202G,376A (G6PD B). Table 3 shows that the small number of females with G6PD202A,376G/202A,376G (G6PD A-) had a trend to lower haemoglobin concentrations but not higher haemolytic component compared to those who were homozygotes for both the G6PD376A and G6PD202G alleles (G6PD B). In contrast, the participants with other combinations of mutant alleles did not have lower haemoglobin concentrations compared to the G6PD B group. Therefore, in further analyses, females who were not homozygotes for both the G6PD376G and G6PD202A alleles were combined into one group for comparison.

Table 3.

Haemoglobin concentration and haemolytic component by different G6PD202,376 genotypes in females. Results are given as mean (SE).

G6PD202G,376A/202G,376A (G6PD B) N=51 G6PD202G,376A/202G,376G or G6PD202G,376G/202G,376G N=56 G6PD202A,376G/202G,376A or G6PD202A,376G/202G,376G N=12 G6PD202A,376G/202A,376G (G6PD A-) N=4
Haemoglobin concentration (g/l), unadjusted 86 (2.0)1 87 (1.5)2 89 (3.2) 80 (11.3)
P* 0.9 0.7 0.3
Haemoglobin concentration (g/l), adjusted** 87 (1.7)1 88 (1.7)2 89 (3.6) 77 (5.4)
P* 0.8 0.7 0.06
Haemolytic component (relative unit), unadjusted 2.9 (0.2)3 2.9 (0.1)4 2.8 (0.2) 2.3 (0.6)
P* 0.9 0.6 0.2
Haemolytic component (relative unit), adjusted*** 2.9 (0.1)3 2.9 (0.1)4 2.8 (0.3) 2.1 (0.4)
P* 0.9 0.8 0.1
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*

Comparison with the G6PD202G,376A/202G,376A (G6PD B) group.

**

Adjusted for recent blood transfusion and haemolytic component

***

Adjusted for hydroxycarbamide treatment and α-thalassaemia

1

N=48

2

N=53

3

N=44

4

N=51

Clinical and laboratory variables according to presence or absence of G6PD202A,376G (G6PD A-) in males and females combined

Clinical characteristics according to G6PD202A,376G category are summarized in Table 4 for all 261 participants. Neither α-thalassaemia genotypes nor Hb F levels varied significantly according to G6PD202A,376G status. Similar results were obtained in analyses restricted to 138 males (data not shown).

Table 4.

Clinical and laboratory variables in children and adolescents with sickle cell anaemia based on G6PD status. Results are median (interquartile range) unless otherwise indicated.

All participants
Demographic variables N Other G6PD genotypes N G6PD202A,376G (G6PD A-) P
Age (years) 238 12 (7–16) 23 12 (9–16) 0.5
Female, N (%) 238 119 (50%) 23 4* (17%) 0.003
Medical history
Hydroxycarbamide treatment, N (%) 234 94 (40%) 23 11 (48%) 0.5
History of chronic transfusion program, N (%) 231 40 (17%) 23 4 (17%) 0.9
>10 units blood transfused life-time, N (%) 228 53 (23%) 23 10 (43%) 0.033
Blood transfusion 58 6 0.3
 past 1 month 29 (50%) 3 (50%)
 past 2 months 16 (28%) 3 (50%)
 past 3 months 13 (22%) 0
Two or more severe pain episodes in last 12 months, N (%) 238 73 (31%) 23 6 (26%) 0.6
Acute chest syndrome history 234 73 (31%) 23 11 (48%) 0.11
History of stroke, N (%) 234 28 (12%) 23 5 (22%) 0.18
Laboratory variables
α-thalassaemia, N.(%) 232 21 0.4
 single deletion HBA1/2 68 (29%) 4 (19%)
 Double deletion HBA1/2 7 (3%) 0 (0%)
Haemoglobin F (%) 123 12 (7–17) 11 7 (3–20) 0.3
Haemoglobin (g/l) 226 86 (77–96) 22 76 (65–90) 0.008
Mean Cell Volume (fl) 225 86 (81–93) 22 88 (81–91) 0.3
White blood cells (109/l) 225 11.5 (8.6–13.7) 22 10.7 (7.7–13.0) 0.6
Erythropoietin (iu/l) 232 70 (46–108) 23 85 (39–140) 0.5
Reticulocyte count (× 109/l) 223 256 (188–351) 21 238 (220–309) 0.3
Aspatate aminotransferase (u/l) 227 45 (33–57) 22 48 (34–60) 0.8
Total bilirubin (μmol/l) 227 42.8 (30.8–63.3) 22 54.7 (37.6–90.6) 0.14
Lactate dehydrogenase (u/l) 218 458 (341–576) 22 432 (315–571) 0.6
Haemolytic component (relative unit) 212 3.1 (2.3–3.7) 21 3.2 (2.7–3.6) 0.6
Tricuspid regurgitation velocity (m/s) 219 2.4 (2.2–2.5) 19 2.3 (2.2–2.5) 0.7
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*

Four females were G6PD202A,376G/202A,376G.

Associations of G6PD202A,376G (G6PD A-) with haemoglobin concentration and markers of haemolysis

In unadjusted analysis, G6PD202A,376G was associated with a lower haemoglobin concentration of 10 g/l on average (P = 0.008) but not with higher values for the haemolytic component (P = 0.6) (Table 4). In multiple linear regression analysis that adjusted for degree of haemolysis, hydroxycarbamide treatment and recent blood transfusion, the association of G6PD202A,376G with lower haemoglobin concentration persisted (Figure 1). There was still no association of G6PD202A,376G with higher haemolytic component after adjusting for α-thalassaemia and hydroxycarbamide therapy (P = 0.7) (Figure 2). Similar analyses for the measurements from which the haemolytic component was derived revealed confirmatory findings: LDH (P = 0.5), AST (P = 0.9), total bilirubin (P = 0.09) and reticulocyte count (P=0.6) were not significantly increased in individuals with G6PD202A,376G (Figure 2).

Figure 1.

Figure 1

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Estimated mean (standard error) haemoglobin concentration in sickle cell anaemia by G6PD202A,376G status. From ANOVA model with adjustment for degree of haemolysis and recent blood transfusion

Figure 2.

Figure 2

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Estimated mean (standard error) haemolytic component, lactate dehydrogense, aspartate aminotransferase, total bilirubin, and reticulocyte count in sickle cell anaemia by G6PD status. From ANOVA model with adjustment for hydroxycarbamide treatment and α-thalasssaemia.

Associations of α-thalassaemia with haemoglobin concentration and the haemolytic component

For comparison, we examined α-thalassaemia, a genetic variation that is known to influence haemoglobin concentration, haemolysis and clinical course in sickle cell anaemia (Billett, et al 1986, Chaar, et al 2006, Embury, et al 1982, Gill, et al 1995, Guasch, et al 1999, Higgs, et al 1982, Mouele, et al 2000, Taylor, et al 2008). In unadjusted analyses, single and double HBA1/2 deletions were associated with progressively higher haemoglobin concentrations (P = 0.005), and these increases in haemoglobin concentration were paralleled by progressively decreasing values for the haemolytic component (P = 0.007). Single HBA1/2 deletion was associated with a 4 g/l increase in haemoglobin concentration and double HBA1/2 deletion with a 10 g/l increase. In multiple linear regression analysis that adjusted for the significant covariates of transfusion in past two months and white blood cell count, the association of HBA1/2 deletions with higher haemoglobin concentrations persisted. After adjustment for these covariates, single HBA1/2 deletion was associated with an estimated 4 g/l increase in haemoglobin concentration and double HBA1/2 deletion with a 6 g/l increase (P for trend = 0.012). Similarly, in multiple regression analysis that adjusted for hydroxycarbamide therapy, the association of HBA1/2 deletions with lower values for the haemolytic component persisted (P for trend = 0.005). Children with α-thalassaemia more commonly had a history of severe pain episodes in the past year compared to children without α-thalassaemia (46% vs. 23%, P < 0.001).

Sub-group of patients not receiving hydroxycarbamide

Among 83 patients not receiving hydroxycarbamide treatment and with no recent blood transfusion, the haemoglobin concentration was lower in participants with G6PD202A,376G (G6PD A-) than the other children in unadjusted analysis (means of 64 vs. 81 g/l, respectively; P = 0.0005) and after adjusting for α-thalassaemia and the haemolytic component (P < 0.0001). Neither the markers of haemolysis nor the haemolytic component were significantly higher in the G6PD202A,376G participants in this subgroup either in unadjusted or adjusted analysis. In this same subgroup, single and double deletion forms of α-thalassaemia were associated with higher haemoglobin concentrations and lower values for the haemolytic component (P for trend <0.001).

Discussion

The present findings suggest that G6PD202A,376G (G6PD A-), as determined by molecular genetic testing, contributes to the degree of anaemia in children and adolescents with sickle cell anaemia by a mechanism that may not include increased haemolysis. Our observation of a reduction in haemoglobin concentration with G6PD202A,376G applied to sickle cell disease children at steady state and was present either in unadjusted analysis or after adjustment for the degree of haemolysis and other important covariates. Likewise, the lack of an association of G6PD202A,376G with haemolysis was observed either in unadjusted analysis or after adjustment for α-thalassaemia, hydroxycarbamide therapy and Hb F.

Most previous studies reported that G6PD A- does not worsen anaemia or increase haemolysis in sickle cell disease (Bernaudin, et al 2008, Gibbs, et al 1980, Steinberg, et al 1988), although occasionally small series have reported a lower haemoglobin concentration with this phenotype (Bouanga, et al 1998). Our present finding that G6PD202A,376G is not associated with increased markers of haemolysis is thus consistent with previous large studies conducted in children (Bernaudin, et al 2008) or in children and adults (Gibbs, et al 1980, Steinberg, et al 1988) with sickle cell anaemia, but the association with lower haemoglobin levels differs from these studies. For instance, the study of Steinberg et al (1998) in children and adults reported that haemoglobin concentrations averaged 4 to 5 g/l lower with G6PD A- compared to G6PD A+ and G6PD B, but this difference was not statistically significant in multivariate analysis (Steinberg, et al 1988). Bernaudin et al (2008), a study in children, reported that the haemoglobin concentration averaged 4 g/l higher with G6PD deficiency, even though G6PD deficiency was significantly associated with increased trans-cranial Doppler velocity, which was linked to more severe anaemia (Bernaudin, et al 2008). An important difference from these previous studies is that we examined a single G6PD deficiency genotype, G6PD202A,376G, rather than the G6PD A-phenotype determined enzymatically, which may include a variety of genotypes in addition to G6PD202A,376G, such as G6PD376G,968C. Also, enzymatic determination of G6PD may have decreased accuracy in hyperhaemolytic states because of higher enzyme concentrations in younger erythrocytes (Beutler, et al 1954, Marks, et al 1958). The facts that we studied the PUSH cohort in a prospective, cross-sectional manner with strictly defined steady state and that we performed G6PD genotyping by molecular genetic testing instead of electrophoresis or enzyme activity may help to explain the variance in the findings with previous studies (Bernaudin, et al 2008, Steinberg, et al 1988) and increase confidence in the present finding of lower haemoglobin concentration with G6PD202A,376G. Furthermore, we performed molecular genetic testing for α-thalassmia in parallel to testing for G6PD202A,376G and observed higher haemoglobin concentrations, less haemolysis, and more frequent pain crises with HBA1/2 deletions. These findings with regard to α-thalassaemia are consistent with the observations of many other investigators (Billett, et al 1986, Chaar, et al 2006, Embury, et al 1982, Gill, et al 1995, Guasch, et al 1999, Higgs, et al 1982, Mouele, et al 2000, Taylor, et al 2008).

There are a number of limitations to our present study. The results are based on a single, cross-sectional analysis rather than on repeated determinations and there was small sample size in the G6PD202A,376G group. Furthermore, we did not perform molecular genetic testing for all of the mutations that could contribute to the G6PD A- phenotype, and we could not rule out G6PD968C in alleles classified as G6PD A in this study.

Given that the haemoglobin concentration was decreased without an apparent increase in haemolysis, our findings raise the possibility that G6PD202A,376G in sickle cell anaemia might be associated with decreased production of erythrocytes. Of note is a study indicating that G6PD plays an important role in erythroid maturation. With complete G6PD deficiency, erythroid precursors are unable to cope with high concentrations of oxygen radicals as haemoglobin synthesis is initiated, and they die by apoptosis (Paglialunga, et al 2004). These results may not be relevant for patients with relatively mild G6PD deficiency associated with G6PD202A,376G. On the other hand, it is conceivable that any slight tendency to increased apoptosis of G6PD202A,376G erythroid precursors at a minimally haemoglobinized stage might become more manifest in the setting of the markedly increased erythropoiesis and hypoxic conditions (low haemoglobin concentration and lower haemoglobin oxygen saturation) of sickle cell anaemia (Aispuru, et al 2008, Qian, et al 2001). Thus, based on our findings, studies of apoptotic markers in early G6PD A- erythroid progenitors exposed to varying degrees of oxidant stress and hypoxia may be of interest. Furthermore, elucidation of the precise pathogenesis of increased anaemia in G6PD-deficient sickle cell anaemia patients may help identify new disease pathways amenable to eventual treatment interventions.

Acknowledgments

Supported in part by grant nos. 2 R25 HL003679-08 and 1 R01 HL079912-02 from NHLBI, by Howard University GCRC grant no 2MOI RR10284-10 from NCRR, NIH, Bethesda, MD, and by the intramural research program of the National Institutes of Health.

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