Co-inheritance of α⁺thalassaemia and sickle trait results in specific effects on haematological parameters

Abstract

Both the sickle cell trait (HbAS) and α⁺thalassaemia are common in many tropical areas. While individually their haematological effects have been well described previously, few studies describe their effects when inherited together. We present data from the coast of Kenya, which suggest that HbAS and α⁺thalassaemia may interact to result in specific effects on haematological parameters. Overall, the difference in Hb concentrations between nonthalassaemics (αα/αα) and α⁺thalassaemia homozygotes (−α/−α) was greater in non-HbAS (HbAA) (0.63g/dl) than in HbAS children (0.25 g/dl). In addition, HbAS ameliorated both the reduced MCV and reduced MCH normally associated with the −α/−α genotype. Potential mechanisms and implications are discussed.

Introduction

The haemoglobinopathies are common throughout much of the tropics. Two of these conditions, sickle cell trait (HbAS) and α⁺thalassaemia, are particularly common in sub-Saharan Africa, having been selected to high frequencies through a survival advantage from malaria. A number of studies have described their individual haematological effects. Most have shown that HbAS has no effect on haemoglobin concentrations (Beutler and West 2005). Similarly, most have shown no effect on MCV, although some suggest that it may result in a mild reduction (Sheehan and Frenkel 1983). On the other hand, both heterozygous (−α/αα) and homozygous (−α/−α) α⁺thalassaemia are associated with moderate reductions in both MCV and Hb (Williams, et al 1996). The cellular pathology of these conditions probably results from an imbalance between α and β-globin chain production, excess β-globin chains forming stable homotetrameric molecules of HbH (β₄) (Rigas, et al 1955), which precipitate as red blood cells age and reduce their longevities.

Despite considerable data, on both HbAS and α⁺thalassaemia individually, little is known about their effects when inherited together. This question is of interest in populations where both conditions are common, and may potentially be relevant to malaria protection in subjects who fall within the various genetic strata. We have therefore investigated haematological indices in children stratified by genotype for HbS and α⁺thalassaemia.

Study Design

Data were provided from participants of a cohort of children, recruited between May 1992 and April 1995, who were subsequently monitored for hospital admission with malaria and other childhood diseases (Snow, et al 2000). Although routine blood sampling was not a part of the original study, between May and October 2000, 2695 surviving members of the original cohort of 3995 were invited to provide a sample for haemoglobin and α⁺thalassaemia genotyping. The KEMRI / National Ethical Review Committee approved the study. Individual written informed consent was provided by all participants or their parents.

Full blood counts were performed on all children using an automated cell counter (MDII; Beckman Coulter, Fullerton, CA). Haemoglobin typing for HbS was conducted by cellulose acetate electrophoresis, and participants were typed for the common African 3.7kb α-globin deletion by PCR (Chong, et al 2000). Between-genotype comparisons were by Student’s t-test and by linear regression, adjusting for the effects of age and sex. Analyses were conducted using STATA version 8.0 (StataCorp, Timberlake, London, United Kingdom).

Results and discussion

Full typing for both HbAS and α⁺thalassaemia were completed on 2141 participants. The biological characteristics and haematological indices of the cohort as a whole and of each genotypic combination separately are summarized in the Table and Figure. In common with previous studies, MCVs were significantly lower in HbAS than HbAA participants. Haemoglobin concentrations were significantly higher, an observation we and others have ascribed to protection from malaria (Desai, et al 2005). However, while the haemoglobin differences across the thalassaemia groups in HbAA children were 0.63g/dl, this drop was only 0.25 g/dl in those with HbAS (Table), suggesting that the haematological effects of α⁺thalassaemia are, to some extent, ameliorated by the co-inheritance of HbAS. This effect on haemoglobin was accompanied by reductions in both microcytosis and hypochromia. Although MCV fell by 9.8fl across the genotypic groups in children with HbAA, it fell by only 7.8fl in children with HbAS. Similarly, MCH fell by 4.1pg in HbAA compared to only 3.3pg in children with HbAS (Table). While the co-existence of β-thalassaemia in this population could potentially alter the interpretation of our observations, no evidence of this condition has been found in previous studies of a representative sample of more than 300 children living in the same area (Yates 1995 and Williams, TN, unpublished observations).

Table.

Haematological indices by haemoglobin phenotype and α⁺thalassaemia genotype

Sickle	α globin	N	Hb (SD)	β (95% CI)	P	MCV (SD)	β (95% CI)	P	RCC (SD)	β (95% CI)	P	MCH (SD)	β (95% CI)	P
AA	All	1828	9.99 (1.75)			74.59 (8.13)			4.17 (0.54)			24.04 (3.47)
AS	All	313	10.27 (1.60)	0.29 (0.08, 0.49)	0.007	72.68 (8.03)	−1.90 (−2.88, −0.93)	<0.001	4.37 (0.53)	0.21 (0.14, 0.27)	<0.001	23.58 (3.27)	−0.46 (−0.87, −0.05)	0.029
AA	αα/αα	637	10.23 (1.82)			78.03 (7.89)			4.01 (0.52)			25.56 (3.48)
AS	αα/αα	116	10.39 (1.50)	0.16 (−0.19, 0.51)	0.361	75.21 (8.37)	−2.82 (−4.40, −1.24)	<0.001	4.23 (0.46)	0.22 (0.12, 0.32)	<0.001	24.69 (3.41)	−0.87 ( −1.56, −0.18)	0.013
AA	–α/αα	879	9.95 (1.75)			74.33 (7.48)			4.17 (0.52)			23.87 (3.15)
AS	–α/αα	151	10.22 (1.79)	0.27 (−0.03, 0.57)	0.081	72.35 (7.44)	−1.97 (−3.27, −0.68)	0.003	4.37 (0.54)	0.19 (0.10, 0.28)	<0.001	23.38 (3.04)	−0.48 ( −1.03, 0.06)	0.079
AA	–α/–α	312	9.59 (1.48)			68.27 (6.22)			4.48 (0.49)			21.42 (2.52)
AS	–α/–α	46	10.15 (1.20)	0.54 (0.10, 1.00)	0.017	67.40 (6.18)	−0.91 (−2.84, 1.02)	0.355	4.75 (0.47)	0.27 (0.12, 0.42)	<0.001	21.43 (2.40)	−0.004 ( −0.78, 0.77)	0.992

The mean age of cohort children at the time of sampling was 7.5 years. 1076/2141 (50.2%) were male, 1030/2141 (48.11%) were heterozygous and 313/2141 (14.6%) homozygous for α⁺thalassaemia while 358/2141 (16.7%) had HbAS. Two children with sickle cell disease (HbSS) were excluded from the analysis. The distribution of genotype combinations (Table) did not differ significantly from that expected on the basis of their individual frequencies.

Figure.

Haematological indices by haemoglobin phenotype and α⁺thalassaemia genotype

Circles AA, triangles AS. Error bars show standard deviations. * p<0.05; ** <0.005.

These observations suggest a biological interaction between HbAS and α⁺thalassaemia and their resultant haematological effects. One well documented interaction is the effect of α⁺thalassaemia on the intra-erythrocytic concentration of HbS in HbAS subjects. Even in non-thalassemic HbAS subjects, the intra-erythrocytic concentration of HbS is lower than that of HbA, probably because of the greater affinity of α–globin for normal (β) than for mutant (β^s) β–globin chains. However, the relative deficiency of α–globin chains in subjects with co-existent α⁺thalassaemia appears to intensify this effect so that, compared to those with both HbAS and αααα, the intra-erythrocytic concentration of HbS is roughly halved in individuals with HbAS and −α/−α (Brittenham, et al 1979, Steinberg, et al 1975). In theory, this might also halve the denaturation and intra-membranous precipitation normally associated with the instability of HbS (Hebbel 2003). Of note in this regard, the decreased intra-erythrocytic concentration of HbS in subjects who co-inherit both sickle cell disease (HbSS) and α⁺thalassaemia is associated with diminished degrees of haemolytic anemia (Embury, et al 1982).

While such considerations might explain how α⁺thalassaemia could reduce the effects of HbS on erythrocyte pathology, they do not explain the converse - how HbS might diminish the pathophysiological effects of α⁺thalassaemia. One possibility is that this relates to the protection that each affords against malaria-induced anemia. As discussed, most have found no association between HbAS and haemoglobin concentration; however, a raised concentration has been found in studies conducted in Africa (Desai, et al 2005), which has been attributed to protection against malaria-induced anemia. We have no data on malaria prevalence in our participants; however, it seems unlikely that our observations are entirely attributable to malaria. First, malaria results in a normochromic, normocytic anemia, and it is not therefore obvious why it should have a differential effect on MCV and MCH in children of differing red cell types. Second, we have recently shown that the malaria protection attributable to both HbAS and α⁺thalassaemia individually is lost when both are inherited together (Williams, et al 2005). As a result, it seems more likely that our observations result from an interaction between HbAS and α⁺thalassaemia at a cellular level.

While the cellular pathology of severe forms of thalassaemia, such as the β-thalassaemias and HbH disease, are well described (Shinar and Rachmilewitz 1993), much less is known about milder forms of α⁺thalassaemia, as described here, or their interactions with other conditions including HbAS. Haemoglobin H inclusions are rarely seen in the red cells of subjects with α⁺thalassaemia, because it believed that most excess β-globin chains are degraded by proteolysis (Wickramasinghe, et al 1984), resulting in a particularly mild phenotype. How this balance is affected by HbAS remains unknown. We suggest that such studies may be relevant to a better understanding of the pathophysiological basis of these conditions, and of their role in malaria resistance.