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Resume Parmi les hemoglobinopathies, deux types de pathologie sont a distinguer. Dans les anomalies de structure, une hemoglobine (Hb) « anormale » est presente, entrainant ou non des signes fonctionnels. L'HbS, responsable de la drepanocytose, y a une place preponderante. Les anomalies de synthese s'expriment dans le groupe tres heterogene des thalassemies. La frequence des hemoglobinopathies en fait un reel probleme de sante publique dans les foyers d'endemie et d'immigration. Les mecanismes physiopathologiques mis en cause sont, cependant, tres differents. La drepanocytose, due a une mutation unique, se presente de facon tres variable, majoritairement comme une maladie rheologique, sa gravite s'etendant de formes letales des l'enfance a d'autres relativement bien tolerees. Pour les thalassemies, l'anemie est au premier plan et tout aussi variable. Des mutations multiples n'expliquent que tres partiellement l'heterogeneite de presentation. Celle-ci, dans les deux cas, fait intervenir l'action de genes modulateurs. Des avances physiopathologiques recentes ont demontre l'implication dans tous les cas de l'endothelium vasculaire, qui se situe au premier plan dans l'evolution d'une drepanocytose. D'autres modulations, multiples, sont progressivement mises en evidence qui modifient l'evolution des thalassemies.

CD1 gene (CD1A to CD1E) products are involved in non-peptide antigen presentation, such as lipids and glycolipids, to T cells. With a similar function to MHC, namely antigen presentation, these genes nevertheless displayed a much lower level of polymorphism as compared to MHC. We report here two additional CD1E variants identified in black African individuals, designated herein CD1E*05 and CD1E*06. While the former differs from the common (wild type) allele sequence by two substitutions at nucleotide positions 217 and 229 of exon 2, the latter only by a single base change at position 91 of exon 3. These substitutions lead to amino acid changes at position 73 and 77 of the alpha1 domain in the former and at position 30 of the alpha2 domain in the latter. Identification of these additional variants suggests that the CD1 locus, especially the CD1E gene, is much more polymorphic than previously assumed.

Resume L'evolution des connaissances a ete explosive ces dernieres annees, concernant les genes de globine, leur regulation normale et les mutations susceptibles de se traduire par une pathologie. Paralleles a ce developpement conceptuel, les progres technologiques permettent de plus en plus souvent de formuler un diagnostic au niveau moleculaire. Une des applications en est le conseil genetique et l'offre d'un diagnostic prenatal. L'etude genotypique peut aussi aider l'interpretation d'un diagnostic difficile. Si la mise en œuvre des techniques de biologie moleculaire est encore du domaine du specialiste, une evolution est previsible, et les principes de ce type de recherche doivent etre connus. Il s'agira d'abord d'extraire l'ADN des cellules nucleees, sang peripherique ou trophoblastes. Deux series de techniques sont d'usage frequent, actuellement normalise: — les techniques de cartographie apres clivage enzymatique mettent en evidence des deletions ou remaniements de l'organisation des genes — la technique d'amplification ciblee par PCR permet, dans une deuxieme etape, une analyse fine et la mise en evidence d'alterations minimes ou ponctuelles. Le choix d'une strategie precise est variable selon les cas. L'evolution tres rapide des connaissances fait que cette strategie, egalement, est susceptible d'evoluer.

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.

CD1 gene (CD1A to CD1E) products are involved in non-peptide antigen presentation, such as lipids and glycolipids, to T cells. With a similar function to MHC, namely antigen presentation, these genes nevertheless displayed a much lower level of polymorphism as compared to MHC. We report here two additional CD1E variants identified in black African individuals, designated herein CD1E*05 and CD1E*06. While the former differs from the common (wild type) allele sequence by two substitutions at nucleotide positions 217 and 229 of exon 2, the latter only by a single base change at position 91 of exon 3. These substitutions lead to amino acid changes at position 73 and 77 of the alpha1 domain in the former and at position 30 of the alpha2 domain in the latter. Identification of these additional variants suggests that the CD1 locus, especially the CD1E gene, is much more polymorphic than previously assumed.

The beta S mutation responsible for sickle cell disease (SCD) was identified in 1949. This mutation always consists in a T for A substitution at codon 6 of the beta-globin chain. Deoxygenated HbS becomes polymerized, producing erythrocyte shape changes and vasoocclusion. Still unexplained is the wide variability in clinical expression of SCD, whose spectrum ranges from severely incapacitating forms to virtually silent forms. Recent research has focused on genetic or environmental factors that may interfere with one or more steps of the basic pathophysiologic mechanisms. These factors can be roughly classified in three groups: i) factors that modify HbS levels within erythrocytes, thereby impacting the propensity of HbS for polymerization; ii) other normal or abnormal hemoglobins present in the erythrocyte that may modify side contacts of the hemoglobin molecule within the polymer, thus either stopping or enhancing polymerization; iii) molecules other than hemoglobin expressed at the surface of the SCD reticulocytes that may adhere to the vascular endothelium, thus slowing flow in the microcirculation and delaying HbS reoxygenation. Whatever the starting point, the same vicious circle is set in motion. These mechanisms have generally been studied separately, in vitro, under artificial conditions. An important goal is to understand how they interact in vivo in a given patient.

Expression of fetal hemoglobin (Hb F) is under polygenic control involving determinants both linked and unlinked to the beta-globin gene cluster on chromosome 11. Variations in the DNase I-hypersensitive site 2 of the locus control region (LCR-HS2) and a C --T change at position -158 from the Ggamma-gene (detected as an XmnI polymorphism) correlate with the high level of Hb F expression in patients with sickle-cell anemia and beta-thalassemia. Interpretation of data under these conditions of anemic stress is difficult because the preferential survival of Hb F-containing erythrocytes (F-cells) may not reflect the true status of Hb F expression. We investigated the relationship between these markers and Hb F expression in terms of F-cell levels in 48 unrelated non-anemic AS heterozygotes from Sicily. The betaS-chromosome of all these individuals was of the Benin haplotype and they differed only by their betaA chromosomes. We demonstrate that F-cell expression is more strongly associated with LCR-HS2 polymorphism than with XmnI polymorphism. The observed association between XmnI polymorphism and Hb F expression is very likely to be due to linkage disequilibrium with LCR-HS2 sequences.

To observe the safety and efficacy of hydroxyurea (HU), a drug that stimulates fetal hemoglobin (Hb F) production, in previously severely ill children with sickle cell disease.HU was given in an uncontrolled study to 35 children with sickle cell disease, aged from 3 to 20 years, suffering from frequent painful crises. Mean duration of treatment was 32 months (range: 12-59 months).HU induced an increase in Hb F levels in all children out one; this increase was maximal after 9 months of treatment, was largely sustained thereafter, and was related to HU dose and inversely to patients' age. We also noted an apparent reduction in crisis, which occurred principally after 3 months of therapy and did not seem strictly correlated with the rise in Hb F level. No serious hematopoietic complication was observed. Growth curves and sexual development were not modified.Our data support the efficacy of HU in reducing painful events in children with sickle cell disease. Short- and middle-term tolerances are good. Thus, we think that HU can be given to children affected by frequent and severe painful crises. We recommend, however, very cautious use of this drug, because its long-term effects in children are still unknown.

Haematological features of 64 patients suffering from non operable cyanotic congenital heart disease (CCHD) treated with hydroxyurea (HU) were compared with those of 43 patients suffering from the same disorder who had not yet received this drug. Patients with subclinical renal dysfunction were excluded by measuring plasma creatinine levels. MCV and HbF were higher among patients receiving HU, the increase in MCV being cumulative with HU dosage but the rise in HbF dose independent. HbF response to HU was found to be due to the coordinated increase in F-cell and F-reticulocyte production rather than to a selective survival of F-cells. Absence of a relationship between plasma erythropoietin and HbF levels excluded a dominant role of the former in increasing F-cell production and results determined after doubling the HU dosage or immediately after initiating therapy suggested genetic differences to be responsible for the individual variations in Hb F response. No irreversible toxic effects or malignancies were noted in this series of patients. HU was administered for a relatively long period of time, the mean duration of treatment exceeding 5 years, while the study also included patients below the age of 10 years.

Results of an epidemiological survey for beta-thalassemic defects involving 239 chromosomes in Algeria are analyzed in relation to the geographic and historical background of the country and are compared with published series for the Tunisian population. Four common mutations account for 81% of the chromosomes, but 13 other defects have been found, illustrating the highly heterogeneous nature of the disease in the northern African countries of the Maghreb. The high frequency of homozygous cases reflects the endogamous social structure of these populations. Distribution of the mutations and linkage to specific RFLP haplotypes provide information concerning their origin and date of introduction in good correlation with the anthropological history of Algeria.

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.

The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5' to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.

A rapid nonradioactive method for the diagnosis of the most frequent Mediterranean beta-thalassemic mutations is described based on a multiplex allele-specific polymerase chain reaction (PCR). This method allows direct detection of normal or mutated alleles on genomic DNA. We have used this approach to detect the most frequent Mediterranean mutations: IVS-1 nt 110 (G----A) and 39 nonsense (C----T). For each mutation three allele-specific oligonucleotides were used: one common upstream primer and two downstream primers differing in their terminal 3' nucleotide (one specific for the normal allele and one for the mutant allele). For each sample two PCR reactions were performed in parallel using in one case IVS-1 nt 110 and codon 39 normal primers and in the second case using the corresponding mutated primers. In both cases the different PCR fragments were visualized. After optimization these primers directed only amplification of their complementary allele. A single blind study was performed on the DNA of 18 individuals who were homozygous or heterozygous for these mutations. In comparison with a parallel investigation, using oligonucleotide probes, all the results were unambiguous. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows the screening of a population group, including heterozygotes, which is required from an epidemiological and anthropological point of view. It could be extended to the large series screening of haplotypes before targeted diagnosis of various genetic diseases.

Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.