[The problem of sickle cell disease in Africa]. / La drépanocytose: problème de l'Afrique.

Sickle cell disease is a genetic blood disorder caused by a glutamic to valine acid substitution in the beta chain of the hemoglobin protein. It was first reported in the United States where most research has been carried out on subjects of African descent. It is diffused throughout the world. Epidemiological data show that the highest incidence of sickle cell anemia is in sub-Saharan Africa where the severest forms are often fatal in children under the age of 5 years. The clinical course of the disease in Africa is comparable to that described in industrialized countries. The three cardinal symptoms are hemolytic anemia, painful episodes, and susceptibility to infection. Genotype and phenotype variations have been observed from one zone to another in Africa. Greater severity is due to a combination of various factors including constant coexistence with Plasmodium falciparum malaria and omnipresence of pyogenic factors as well as to the unfavorable demographic setting involving endogamy, poor healthcare facilities, and poor socio-economic conditions. A hundred years of research has provided a good understanding of the pathophysiological mechanisms that can sometimes be improved by to primary hydroxyurea therapy. Sickle cell disease remains a major health problem in Africa where patients do not currently benefit from the same treatment as in industrial countries.

A gene conversion located 5' to the A gamma gene in linkage disequilibrium with the Bantu haplotype in sickle cell anemia.

Cloning and sequencing of the gamma-globin gene of a sickle cell anemia patient homozygous for the Bantu haplotype has revealed a gene conversion that involves the replacement of an A gamma sequence by a G gamma sequence in the promoter area of the A gamma gene. This event is similar to another gene conversion believed to be responsible for the very high homology between gamma-globin genes, suggesting that the promoter area of these genes is prone to this type of genetic rearrangement. Further analysis demonstrated that the chromosome bearing this gene conversion has a very high frequency among Bantu chromosomes and a very low or nil frequency in other haplotypes linked to the beta s gene. No correlation was found between the G gamma/A gamma ratio and the presence of the gene conversion among Bantu haplotype patients, thus excluding a portion of the gamma gene sequence in the determination of this ratio.

Sickle gene. Its origin and diffusion from West Africa.

Linked DNA polymorphisms can be used to study the evolution of structural gene mutations. Both the beta S-(beta 6Glu leads to Val) and beta C-(beta 6Glu leads to Lys) genes are common in West Africa. We have analyzed their linkage to a polymorphic Hpa 1 site appearing 3' to the beta-globin gene locus in selected populations from Wes Africa. A large reservoir of beta A-genes linked to 13-kilobase Hpa 1 fragments with a frequency of 17-18% has been identified. In addition, the beta S- and beta C-genes in Togo are found to be tightly linked to the 13-kilobase Hpa 1 fragment, whereas 72% of the beta S-genes in the Ivory Coast reside on the 7.6-kilobase Hpa 1 fragment. These studies are consistent with the selection and expansion of two different chromosomes bearing beta S-genes in at least two physically close, but ethnically separate regions of West Africa, with subsequent diffusion to North, Equatorial, and East Africa.

Impairment of the growth of Plasmodium falciparum in HbEE erythrocytes.

HbE is a beta-chain mutant frequently found among inhabitants of Southeast Asia and surrounding territories. We find that Plasmodium falciparum multiplies more slowly in erythrocytes from individuals homozygous for HbE than in cells from HbA individuals. In contrast, this parasite grows normally in erythrocytes heterozygous for HbE. This is the first direct evidence that suggests what has been suspected on the basis of circumstantial data, that HbE-containing erythrocytes might be advantageous to the carrier in regions with endemic malaria.

Hemoglobin Cochin-Port-Royal: consequences of the replacement of the beta chain C-terminal by an arginine.

Hemoglobin Cochin Port-Royal beta 146 (HC3) His yields Arg is the second example in which the beta C-terminal residue is replaced. Owing to the known importance of His beta 146 in the co-operative effects of hemoglobin, the functional properties of this variant were carefully studied. It had a normal Hill coefficient but a reduced alkaline Bohr effect. However, the reduction in Bohr effect is less than the halving predicted from previous mutants and modified hemoglobins.

Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala: isolation and functional properties of the heme saturated form.

Hemoglobin Djelfa beta98 (FG 5) Val leads to Ala is a neutrally substituted unstable hemoglobin, exhibiting the same gross features as hemoglobin Köln beta98 (FG 5) Val leads to Met. In addition to the presence of a deheminized fraction, a heme saturated abnormal hemoglobin was visualized and isolated by high resolution electrofocusing. By functional studies of the fully heminized form, a slightly increased oxygen affinity, an impairment of heme-heme interaction and a decreased response to organic phosphates were demonstrated. These functional perturbations point out the importance of the beta98 invariant valyl residue, in the quaternary contacts. They can account for the poor oxygen delivery of erythrocytes.

Structure and function of Hb Saint-Jacques (alpha 2 beta 2 140 (H18) Ala----Thr): a new high-oxygen-affinity variant with altered bisphosphoglycerate binding.

A low P50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g X dl-1). Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate. Isoelectrofocusing (pH gradient 6-8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples. Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG). Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates. Structural studies were carried out on the mixture of beta A + beta X chains and revealed the presence of two beta Tp14 peptides. Sequencing the abnormal beta Tp14 peptide showed the substitution Ala----Thr of the beta 140 (H18) residue. This new variant was named Hb Saint-Jacques. Examination of the three dimensional model of HbAo indicates that the substitution beta 140 (H18) Ala----Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the beta chains. This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two beta chains. Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.

Influence of thyroid status on hemoglobin A2 expression.

We studied the electrophoretic pattern of hemoglobin (Hb) and red blood cell indices in 128 women divided into four groups: group I, 36 nonanemic hyperthyroid women, divided in two subgroups: 36 with untreated hyperthyroidism (subgroup IA) and 9 made euthyroid by antithyroid drug therapy (subgroup IB); group II, 12 nonanemic women with untreated hypothyroidism; group III, 30 women known to be heterozygous for beta-thalassemia; and group IV, 50 healthy women. The mean (+/- SEM) HbA2 level was higher (P less than 0.001) in subgroup IA (3.21 +/- 0.06%) than in subgroup IB (2.42 +/- 0.09%) and group IV (2.48 +/- 0.04%), but lower (P less than 0.001) than in group III (5.26 +/- 0.12%). The mean HbA2 level was lower (P less than 0.001) in group II (1.99 +/- 0.08%) than in group IV. Hb fetal was detectable in eight patients of subgroup IA and undetectable in subgroup IB and groups II and IV. The mean cellular volume was lower (P less than 0.001) in subgroup IA than in other nonanemic groups. The mean cellular volume was higher (P less than 0.001) in group II than in group IV. Follow-up of nine patients who became euthyroid with treatment showed the normalization of these erythrocyte parameters. These results suggest that thyroid hormones can modulate the synthesis of delta- and gamma-globin chains.

Haemoglobin D-Ouled Rabah among the Mozabites: a relevant variant to trace the origin of Berber-speaking populations.

We have studied haemoglobin (Hb) variants and blood groups (ABO, RH, and Kell) in 598 children from the Berber population of the Mzab. Hb D-Ouled Rabah, considered as a private marker of the Kel Kummer Tuaregs, and Hb C were found at the same gene frequency (0.015). Haplotype analysis suggests a single origin to the Hb D mutation. Genetic distances calculated from the blood group data cluster Mozabites and Tuaregs with the other Berber-speaking groups, Arabic-speaking populations being more distant. But, we found no specific relationship between Mozabites and Kel Kummers. Tuaregs in general exhibit features that tend to differentiate them from other Berber-speaking groups. Hb D-Ouled Rabah may be specific of Berber-speaking populations.

Diversity of sequence haplotypes associated with beta-thalassaemia mutations in Algeria: implications for their origin.

We report the allelic sequence polymorphism associated with seven beta-thalassaemia mutations. Thirty-two DNAs originating from Algeria and 12 DNAs from Sardinia and Sicily were investigated. Their analysis revealed an association with a unique haplotype for three beta-thalassaemia mutations (-29, IVS-I-2 and IVS-I-1). It seems clear that these mutations have a unicentric origin. The presence of the -29 mutation could be explained by migration and founding effect. However, the local origin of IVS-I-2 seems clear. The four other mutations, FS6, IVS-I-6, IVS-I-110 and stop39 were found to be associated with at least two different sequence haplotypes. The likelihood of so many recurrent nucleotide dimorphisms in different lineages as a consequence of random mutation is very low; it is supported neither by the analysis of equivalent regions in other primates, nor by the presence of highly mutable sites such as CpG dinucleotides. The fact that these mutations are found exclusively in the Mediterranean area is not in favour of a recurrent origin of the mutation. The diversity is far more important for the preponderant thalassaemia mutations of the Mediterranean area and is higher in the 5' part of the beta-globin gene. Hence, the IVS-I-110, the preponderant beta-thalassaemia in the Eastern Mediterranean, probably emerged in the extension of the fertile crescent. For the stop39, all the data support the hypothesis of a West-Mediterranean origin. The diversity of haplotypes would then be generated by recombination events (crossing-over or gene conversions) between the original beta-thalassaemia chromosome and the other chomosomal structures present in the normal population.

Abnormal hemoglobin synthesis in some leukemic patients.

Hemoglobin chain synthesis during leukemic processes has been studied on patients having fetal hemoglobin. All cases showed the following abnormalities : (1) a relatively increased synthesis of the beta chain ; (2) an important increase of the free dimeric precursors pool, with, most of the time, a predominance of alpha chain. If the first point suggests an alpha-thalassemia feature, the presence of free alpha chains shows evidence for a more complex mechanism not only due to a decrease of messenger RNA. The hypothesis of a clonal disorder could neither be demonstrated nor ruled out. The observed abnormalities could be due to a defect in a alpha chain depending regulation mechanism.

Ca++ binding properties of human prothrombin.

The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin.

Isolation of preliminary characterization of a vitamin K dependent peptide from human prothrombin.

A peptide containing the vitamin K dependent Ca2+ binding region of human prothrombin has been isolated by adsorption of the tryptic digest of the native protein on barium citrate. Its aminoacid composition has been determined. The N- and C-terminal residues have been characterized. The obtained results are showing some slight differences with the corresponding bovine peptide. Since they were not strictly identical for two different preparations, the eventuality of a molecular heterogeneity is discussed.

Quantitation of hemoglobin A1c: a rapid, automated precision-chromatography technique.

Hemoglobin A1c is increased in patients with diabetes mellitus and its level reflects the status of blood glucose equilibrium over a period of several weeks. The practical use of its estimation was hampered by technical difficulties in investigating large series of samples. In order to apply this examination for routine purposes we describe in this paper acceleration and full automatization of the original chromatographic method allowing quantitation of hemoglobin A1c in 45 min.

Isoelectrofocusing: a method of multiple applications for hemoglobin studies.

A microanalytical screening of hemoglobins by isoelectric focusing on acryl-amide gel columns is described. Addition of this technique to our regular analytical procedures revealed many mutants, praticularly some neutral ones which were not resolved by other conventional procedures. Extension of this technique to a quantitative scale permitted rapid isolation of some unstable mutants in an almost pure form ready for functional studies. The natural color of the separated components is often indicative of their nature and in cases like the sulfhemoglobin, Hbs M, they are of diagnostic value. The heme-losing, abnormal, unstable mutants, on the addition of external cyanhemin, take them up and change their isoelectric pH. This is an attractive visible procedure for their diagnosis. From all these results, it appears that isoelectric focusing as a single method brings more information than the other conventional procedures and hence can be considered as a semi-routine investigation for hemoglobin studies before deciding on a much longer procedure.

[Recent data concerning abnormal hemoglobins (author's transl)]. / Données récentes sur les hémoglobines anormales.

The study of abnormal hemoglobins was oriented towards three criteria as a function of commonly encountered clinical problems : differentiation between frequent, relatively infrequent and exceptional hemoglobins ; systematic study of high-risk ethnic groups and isolated discoveries ; silent hemoglobinopathies and those accompanied by physiopathological repercussions such as hemolytic anemia, disturbance in oxygen transport and cyanosis. The chronology and rationale for these studies was discussed, with particular attention being given both to normalized baseline measures and to the new, high-resolution techniques such as isoelectric focalization. No attempt was made to provide an exhaustive list, but rather examples were provided illustrating the various points considered. Finally, a number of particular points are brought up to where a correctly made diagnosis can avoid the necessity of subjecting patients to long and possibly dangerous tests.

[Intermediate homozygous beta-thalassemia with high fetal hemoglobin synthesis. Molecular analysis]. / Bêtathalassémie homozygote intermédiaire avec synthèse élevée d'hémoglobine foetale. Analyse moléculaire.

The intermediate character of beta-thalassaemia may be due to a molecular cause capable of reducing the unbalance between alpha and non-alpha chains. We report the case of a child born of consanguineous parents and homozygous for beta-thalassaemia. The patient was irregularly transfused until puberty. His haemoglobin level was around 10 g/dl, and in his erythroid line only foetal haemoglobin was synthesized, except for small amounts of haemoglobin A2. An in vitro study of globin chains biosynthesis confirmed the total absence of beta chains synthesis, and the molecular defect was characterized. This was deletion of a colon 6 nucleotide on both chromosomes, making the messenger RNA unstable and non-traducible. The initial diagnostic approach in this patient included the indirect determination of restriction polymorphism (Orkin's halotype IX), a search for the presence or absence of a nonsense codon 39 often associated with haplotype IX, and the demonstration of a frameshift in the reading phase of codon 6. The very high synthesis of foetal haemoglobin in this patient seems to be linked with a C----T substitution in -158 of the G gamma gene creating an Xmnl site and a gamma phi beta (+-++) subhaplotype which appears to be related in all haemoglobinopathies to an increased synthesis of foetal haemoglobin with predominant G gamma chain.

[Homozygous beta-thalassemia in Algeria. Study of 50 cases]. / La beta-thalassémie homozygote en Algérie. Etude de cinquante cas.

The clinical and laboratory data recorded at first presentation in 50 homozygous beta-thalassaemic untransfused children seen at the National Transfusion Centre, Algiers, are reported. These children came from 38 families, including 25 with consanguinous parents. Pallor was observed in all cases but jaundice and asthenia were present in only 11 and 10 children respectively. Splenomegaly was frequent (45 cases), as were skeletal changes mainly in the skull and face (35 cases). Haemoglobin levels ranged from 2.4 to 9.6 g/dl and MCV from 71 to 89 fl. Among these 50 patients, 34 had beta + thalassaemia and 16 beta 0 thalassaemia. Levels of foetal haemoglobin (Hb F) were similar in both groups but clinical symptoms appeared earlier in beta 0 thalassaemia patients. Thirty-seven cases were diagnosed as thalassaemia major and 6 as thalassaemia intermedia. Comparison of various parameters between siblings (20 children belonging to 9 families) showed no differences between Hb F and Hb A2 levels and clinical courses. These findings should be taken into consideration for the prenatal diagnosis of beta-thalassaemia.

[Treatment of homozygote beta thalassemia in Algiers. A 5-year follow-up of 66 patients]. / Traitement des beta-thalassémies homozygotes à Alger. Suivi de 66 malades sur 5 ans.

During the years 1982-1987, 66 patients with homozygous beta-thalassaemia were treated at the blood transfusion centre of Algiers. The patients, aged from 1 to 23 years in 1982, came from 48 families, 30 of which were issued from consanguinous unions. The patients fell into three groups according to the early institution and quality of treatment (blood transfusions, antibiotic therapy, desferrioxamine given when available). The beneficial clinical effects observed (satisfactory growth and development, reduction of splenomegaly and hypersplenism, attenuation of craniofacial malformations, performance at school) seemed to be directly related to the mean haemoglobin level prior to transfusion and to the early institution of treatment. Four patients died of anaemia and haemochromatosis. The incidence of viral contamination was 27.5 per cent for the hepatitis B virus and nil for the human immunodeficiency virus.

[Genetic aspects of sickle cell anemia]. / Historie génétique de la drépanocytose.

The genetics of sickle cell anemia may be considered as a model. Its mendelian transmission was hypothesized even before the molecular era. Once the mutation identified, it could be studied at the protein and DNA level; a consistent pathophysiological mechanism was proposed; the various genetic forms of the disease could be identified; the way by which a balanced polymorphism with Plasmodium falciparum malaria is obtained was analyzed. More recently, investigations were run in order to understand how modulating, or epistatic factors could modify the pathophysiological mechanism and contribute to the high clinical diversity of the disease. Several factors have been identified, among which a concomitant alpha-thalassemia, an overproduction of fetal hemoglobin, due either to an activation of the gamma genes or to an increase of the F-cell number, and finally a quantitative control of the beta s chains themselves. Such a high number of genetic active factors questions the concept itself of a monogenic disease.