Increased Microerythrocyte Count in Homozygous ��+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia

Background

The heritable haemoglobinopathy α⁺-thalassaemia is caused by the reduced synthesis of α-globin chains that form part of normal adult haemoglobin (Hb). Individuals homozygous for α⁺-thalassaemia have microcytosis and an increased erythrocyte count. α⁺-Thalassaemia homozygosity confers considerable protection against severe malaria, including severe malarial anaemia (SMA) (Hb concentration < 50 g/l), but does not influence parasite count. We tested the hypothesis that the erythrocyte indices associated with α⁺-thalassaemia homozygosity provide a haematological benefit during acute malaria.

Methods and Findings

Data from children living on the north coast of Papua New Guinea who had participated in a case-control study of the protection afforded by α⁺-thalassaemia against severe malaria were reanalysed to assess the genotype-specific reduction in erythrocyte count and Hb levels associated with acute malarial disease. We observed a reduction in median erythrocyte count of ∼1.5 × 10¹²/l in all children with acute falciparum malaria relative to values in community children (p < 0.001). We developed a simple mathematical model of the linear relationship between Hb concentration and erythrocyte count. This model predicted that children homozygous for α⁺-thalassaemia lose less Hb than children of normal genotype for a reduction in erythrocyte count of >1.1 × 10¹²/l as a result of the reduced mean cell Hb in homozygous α⁺-thalassaemia. In addition, children homozygous for α⁺-thalassaemia require a 10% greater reduction in erythrocyte count than children of normal genotype (p = 0.02) for Hb concentration to fall to 50 g/l, the cutoff for SMA. We estimated that the haematological profile in children homozygous for α⁺-thalassaemia reduces the risk of SMA during acute malaria compared to children of normal genotype (relative risk 0.52; 95% confidence interval [CI] 0.24–1.12, p = 0.09).

Conclusions

The increased erythrocyte count and microcytosis in children homozygous for α⁺-thalassaemia may contribute substantially to their protection against SMA. A lower concentration of Hb per erythrocyte and a larger population of erythrocytes may be a biologically advantageous strategy against the significant reduction in erythrocyte count that occurs during acute infection with the malaria parasite Plasmodium falciparum. This haematological profile may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage.     

Thalassaemia in the British

Different forms of thalassaemia or related disorders were found in 116 people of apparently pure British stock. Among them were one family with a child homozygous for β-thalassaemia and eight heterozygous relatives, 16 families with 83 persons heterozygous for β-thalassaemia, two families with three persons with Hb H disease and three heterozygous for α-thalassaemia 1, one family with a child apparently homozygous for the “silent β-thalassaemia gene,” one family with six members heterozygous for a form of β-thalassaemia intermedia, and three families with 11 members heterozygous for different types of hereditary persistence of fetal haemoglobin. The clinical, haematological, and haemoglobin biosynthetic findings in these persons were similar to those of patients with thalassaemia from other racial groups. The heterozygous state for β-thalassaemia is overlooked in British patients, particularly during pregnancy, because it is not considered in the differential diagnosis of refractory anaemia. In many cases this leads to much unnecessary investigation and potentially harmful treatment.There seem to be several varieties of hereditary persistence of fetal haemoglobin production among British people. These conditions, while not causing anaemia, may cause difficulties during examination of maternal blood for fetal cells and may, if inherited with a β-thalassaemia gene, produce an unusually high level of Hb F in a person heterozygous for β-thalassaemia.     

Benign Obstetric History in Women with Sickle-cell Anaemia Associated with α-Thalassaemia

Two Ghanaian women with sickle-cell anaemia and α-thalassaemia were found to have an unusually benign obstetric history. In addition to two factors present which are known to moderate the clinical course of sickle-cell anaemia, good socioeconomic status and a relatively high Hb F level, it is suggested that α-thalassaemia may act among other things by lowering the haemoglobin concentration in the red cells and thereby lowering their tendency to sickle in vivo.     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.     

Increased susceptibility of malaria-infected variant erythrocytes to the mononuclear phagocyte system

The interactions of the mononuclear phagocyte system with Plasmodium falciparum-infected genetically variant erythrocytes may result in a significant protection for the host. Infected hemoglobin (Hb) EE and Hb EA erythrocytes are more susceptible to phagocytosis by monocytes than are infected Hb AA erythrocytes. The increased susceptibility to phagocytosis of infected erythrocytes was also found for a number of genetic variants involving the alpha-globin chain, namely, alpha-thal 1 trait (--/alpha alpha), alpha-thal 2 trait (-alpha/alpha alpha), Hb H (--/-alpha), Hb H/Hb Constant Spring (CS) (--/alpha CS alpha), Hb CS trait, and homozygous Hb CS erythrocytes. In addition, oxidative damage from hydrogen peroxide, produced in simulation of macrophages, led to much more effective killing of parasites in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes than in normal ones. Parasites infecting Hb H/Hb CS also showed an enhanced sensitivity to hydrogen peroxide.     

Haemolytic anaemia in Basenji dogs. 2. Partial deficiency of erythrocyte pyruvate kinase (PK; EC 2.7.1.40) in heterozygous carriers1

Congenital nonspherocytic haemolytic anaemia (HA) in dogs of the Basenji breed is inherited as a simple, autosomal recessive trait. Previous results of pyruvate kinase (PK) assays suggest a causal relationship between the anaemia and PK deficiency in erythrocytes. In the present investigation assays of this enzyme have been performed on haemolysates from 45 Basenji dogs, comprising 3 anaemic and 42 non-anaemic individuals of which 13 were known heterozygotes. The PK activity in haemolysates from the 42 non-anaemic dogs exhibited a bimodal distribution corresponding to the genotypic classes: heterozygotes and normal homozygotes. The results indicate that heterozygotes have a partial, detectable enzyme deficiency, not reflected in clinical disease, and thus give evidence of a gene dosage effect in agreement with observations in man. The proposed genotypes PK PK, PK pk and pk pk refer to normal homozygotes, heterozygotes, and anaemic individuals, respectively. The findings strengthen the genetic hypothesis of recessiveness of the anaemia by direct detection of heterozygosity of parents of affected individuals. Moreover, the results are of value in comparative studies and they have practical application in connection with eradication programmes.     

Sickle cell-beta 0-thalassaemia in Saudi Arabia

During an extensive investigation to determine the frequency of sickle cell and thalassaemia genes in the Saudi population, 22 cases with S/beta 0-thalassaemia were identified and the haematological, biochemical and clinical findings were compared with those in patients with sickle cell anaemia. The values of mean cell volume, mean cell haemoglobin and packed cell volume were found to be lower while all other haematological parameters including Hb A2 were higher in the S/beta 0-thalassaemia group. No statistically significant difference in the Hb F level was found between the two groups. Biochemical parameters were grouped according to organ function tests. Only slight differences were seen in the values of some parameters. The clinical data showed that, in general, patients with sickle cell anaemia had a more severe condition than the S/beta 0-thalassaemia.     

Antibodies to actin in autoimmune haemolytic anaemia

Background  

In autoimmune haemolytic anaemia (AIHA), autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity.     

Expression of Hb β-T and Hb β-E genes in Eastern India—Family studies

The distribution patterns of different haemoglobins were observed amongst the family members of β-thalassaemia homozygous and HbE-β-thalassaemia patients with the aid of gel electrophoretic and alkali denaturation techniques. Of the 18 families studied, four belonged to β-thalassaemia homozygous and 14 to HbE-β-thalassaemia patients. Interaction of HbE and β-thalassaemia genes resulted in major clinical abnormalities with increase in the percentages of haemoglobins F and E. The percentages of HbA₂ in homozygous β-thalassaemia were within the normal range. Although in Southeast Asia the β° type of HbE-thalassaemia is more prevalent, only one individual with this type of thalassaemia was observed during this survey. In the rest of the patients examined the percentages of adult haemoglobin ranged from 5.2 to 42.5 indicating the presence of a β⁺ type gene.     

α-Thalassaemia in Tunisia: some epidemiological and molecular data

Unlike the other haemoglobinopathies, few researches have been published concerning α-thalassaemia in Tunisia. The aim of the present work is to acquire further data concerning α-thalassaemia prevalence and molecular defects spectrum in Tunisia, by collecting and studying several kinds of samples carrying α-thalassaemia. The first survey conducted on 529 cord blood samples using cellulose acetate electrophoresis, have displayed the prevalence of 7.38% Hb Bart’s carriers at birth. Molecular analyses were conducted by PCR and DNA sequencing on 20 families’ cases from the above survey carrying the Hb Bart’s at birth and on 10 Hb H diseased patients. The results showed six α-globin gene molecular defects and were responsible for α-thalassaemia: -α^3.7, - -^MedI, α^TSaudi, α₂^{cd23GAG→Stop}, Hb Greone Hart: α₁^119CCT→TCT corresponding to 11 genotypes out of which two are responsible for Hb H disease (- -^Med/-α^3.7) and (α^TSaudiα/α^TSaudiα) and a newly described polymorphism: α^+6C→G. The geographical repartition of α-thal carriers showed that the -α^3.7 deletion is distributed all over the country, respectively the α^HphI and α^TSaudi seem to be more frequent in the central region of the northeast region. The haematological and clinical data showed a moderate phenotype with a late age of diagnosis for Hb H disease. This work had permitted, in addition to an overview on α-thalassaemia in the country, the optimization of protocols for α-thalassaemia detection in our lab, allowing further investigations concerning phenotype-genotype correlation in sickle cell disease or β-thalassaemia.     

A novel role for acid‐sensing ion channels in Pavlovian reward conditioning

Pavlovian fear conditioning has been shown to depend on acid‐sensing ion channel‐1A (ASIC1A); however, it is unknown whether conditioning to rewarding stimuli also depends on ASIC1A. Here, we tested the hypothesis that ASIC1A contributes to Pavlovian conditioning to a non‐drug reward. We found effects of ASIC1A disruption depended on the relationship between the conditional stimulus (CS) and the unconditional stimulus (US), which was varied between five experiments. In experiment 1, when the CS preceded the US signaling an upcoming reward, Asic1a^?/? mice exhibited a deficit in conditioning compared to Asic1a^+/+ mice. Alternatively, in experiment 2, when the CS coinitiated with the US and signaled immediate reward availability, the Asic1a^?/? mice exhibited an increase in conditioned responses compared to Asic1a^+/+ mice, which contrasted with the deficits in the first experiment. Furthermore, in experiments 3 and 4, when the CS partially overlapped in time with the US, or the CS was shortened and coinitiated with the US, the Asic1a^?/? mice did not differ from control mice. The contrasting outcomes were likely because of differences in conditioning because in experiment 5 neither the Asic1a^?/? nor Asic1a^+/+ mice acquired conditioned responses when the CS and US were explicitly unpaired. Taken together, these results suggest that the effects of ASIC1A disruption on reward conditioning depend on the temporal relationship between the CS and US. Furthermore, these results suggest that ASIC1A plays a critical, yet nuanced role in Pavlovian conditioning. More research will be needed to deconstruct the roles of ASIC1A in these fundamental forms of learning and memory.     

Chlorsulfuron resistance in Daucus carota cell lines and plants:Involvement of gene amplification

Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level.     

Starch-gel electrophoretic variants of erythrocyte 6-phosphogluconate dehydrogenase in asian macaques

Five alleles with eight electrophoretic phenotypes of 6-phosphogluconate dehydrogenase were found in 1,195 blood samples from fourteen populations of nine macaque species.Macaca fascicularis from Malaya showed the most polymorphism, with three Pgd alleles resulting in five phenotypes.Macaca mulatta, M. speciosa, M. nemestrina, andM. cyclopis had two alleles each (although the last two species showed a high percentage of homozygosity). The remaining four species (M. fuscata, M. radiata, M. maura, andM. nigra) were homozygous for the Pgd^a allele. The predominance of Pgd^a was observed in all macaque species, exceptM. speciosa which showed a high (57%) frequency of Pgd^d. The distinctive position ofM. speciosa with regard to 6PGD variants parallels observations that indicate that this species carries transferrin and carbonic anhydrase I alleles in different frequencies from those of the other macaque species. Other similarities between the patterns of transferrin and 6PGD variations include a tendency toward homozygosity at the Pgd locus in the insular macaque forms. However, in this case only the Pgd^a allele is involved, while some variation was found in the transferrin alleles fixed by the founder effect in the insular macaques.This research was supported by NSF grants GF 253, GB 7426, and GB 15060 of the U.S.-Japan Cooperative Science and Systematic Biology Programs.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

Human leucocyte antigens A,B, C,and DRW in idiopathic "warm" autoimmune haemolytic anaemia.

Twenty patients with idiopathic "warm" autoimmune haemolytic anaemia and 40 controls were types concurrently for human leucocyte antigens (HLA) A, B, C, and DRW. There was a significantly stronger association of HLA-B8 with the disease (chi 2 = 10.39; p = 0.018) than HLA-DRW3 (chi 2 = 3.71; P = 0.35) and the patients also showed a significant increase in BW6 homozygosity (chi 2 = 7.13; P = 0.01) and a corresponding reduction in BW4 (chi 2 = 7.13; P = 0.02). (All p values corrected for number of antigens at each locus.) These findings suggest that susceptibility to idiopathic autoimmune haemolytic anaemia is associated more closely with the HLA-B locus than with DRW3.     

Improved detection of β-thalassaemia carriers by a two-test method

Summary Seven red cell parameters, taken one at a time and in their 21 possible pairs, were investigated for their power to discriminate between adult carriers of the -thalassaemia allele and adult normal subjects. The red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), and haemoglobin A₂(HbA₂) fraction were measured in 24 obligate heterozygotes and in 99 adult controls with comparable age and sex distributions. Quadratic discriminant functions were computed using Bayesian analysis of univariate and bivariate Gaussian density functions. Classification errors were then calculated by integrating the density function for one genotype over the region assigned to the other.In the univariate case, MCH led to the lowest cost of misclassification while MCV was the second best discriminant for all posterior probabilities considered. In the bivariate case, MCV combined with percentage Hb A₂ yielded the best discrimination and generated misclassification costs roughly 1/30 of those generated by the most efficient single parameter. When use of MCV alone cannot classify an individual reliably either as a heterozygote or as homozygous normal, combined use of MCV and percentage Hb A₂ is recommended for maximum accuracy.Application of this screening method to 260 adult subjects at risk for thalassaemia heterozygosity yielded an unbiased frequency of 0.067 for the adult carrier in the Montreal Greek community, a value similar to that reported in the source population in Greece. The improved discriminations thus achieved is particularly useful for sibs of affected subjects whose high prior probability of heterozygosity (0.67) impairs classification.     

Anaemia,Zinc and Copper Deficiencies Among Pregnant Women in Central Sudan

Anaemia is a widespread problem in many parts of the world especially in tropic areas. Among pregnant women, it has negative consequences on maternal and perinatal outcomes. A cross-sectional study was conducted to investigate the prevalence of anaemia, iron, zinc and copper deficiencies among pregnant women in Wad Medani hospital, central Sudan and to examine the relationship of these micronutrients with haemoglobin (Hb) levels. One hundred four (52.5%) out of 200 pregnant women had anaemia (Hb < 11 gm/dl) and 3 (1.5) % had severe anaemia (Hb < 7 gm/dl). Iron deficiency (S-ferritin < 15 μg/l), iron deficiency anaemia (<11 gm/dl and S-ferritin < 15 μg/l) were prevalent in 25 (12.5%) and 13 (6.5%) of these women, respectively. Ninety (45.0%) and eight (4.0%) of these women had zinc (<80 μg/ml) and copper (<80 μg/ml) deficiency, respectively. In 24 (12.0%) of these women, there were ≥2 deficiencies of these elements. S-copper was significantly lower in patients with anaemia. While age, parity, gestational age, ferritin, zinc and copper were not predictors for anaemia, women who practiced pica were at higher risk for anaemia (OR = 3.4, 95% CI = 1.4–7.9, P = 0.004). Gestational age was significantly inversely correlated with haemoglobin (r = 0.161, P = 0.03), S-ferritin (r = 0.285, P = 0.001) and S-zinc (r = 0.166, P = 0.02). Thus, dietary and supplement interventions are required to prevent and control anaemia in this setting. Further research is needed.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon

Monoecy is an important goal for melon breeding because of the agronomic advantages it provides to parental lines in that they do not require hand emasculation to develop monoecious F₁ hybrids, the latter producing fruits of higher quality. Monoecious phenotype is conferred by the dominant allele of the andromonoecious (a) gene, whereas recessive homozygous plants are andromonoecious. A bulked segregant analysis (BSA) approach performed in a set of 38 double-haploid lines has allowed us to identify an AFLP marker linked to the a gene at 3.3 cM. Following cloning and sequencing of the AFLP fragment, specific PCR primers were designed and used in the amplification of a codominant SCAR marker. Using a backcrossed mapping population of 530 plants, the SCAR marker could be mapped near the a locus (5.5 cM). Size difference between the two allelic SCAR fragments is 42 bp and might be due to a deletion/insertion. The SCAR marker is closest to the a gene identified to date, and can be useful in breeding programs, using marker-assisted selection procedures to screen for sexual types in melon.     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.