Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee.

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.

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.

The spectrum of beta-thalassemia mutations in the Oran region of Algeria.

In order to delineate the spectrum of beta-globin gene defects causing beta-thalassemia in the Oran region of Algeria, we have analyzed a representative sample of 31 beta-thalassemia patients. This led to the detection of 10 mutations. Four of them [nonsense codon 39 (C->T), IVS-I-110 (G->A), IVS-I-2 (T->C), and frameshift codon 6 (-A)] account for approximately 77% of the beta-thalassemia chromosomes. Three of these mutants are also widespread in Mediterranean populations, whereas the fourth, IVS-I-2 (T->C), appears typical of the Oranese population. The six other variants are less frequent. The possible origin of these mutated alleles, either by recurrent mutational event or by migration from other populations, is discussed.

[Abnormal hemoglobins in Algeria]. / Hémoglobines anormales en Algérie.

The existence of frequent hemoglobin abnormalities raises a Public Health problem in Algeria. The presence of genes for hemoglobin S, hemoglobin C and thalassemia in various regions of the country causes severe congenital hemolytic anemias: thalassemia, sickle-cell anemia, S thalassemia, association of HbS and HbC. These diseases are often invaliding requiring frequent admissions to hospital and have a high social cost. We have also observed certain rare hemoglobins, of which some, hemoglobin Setif, hemoglobin D Ouled Rabah were described for the first time in Algerians. They are not pathogenic in the heterozygous state.

Genetic and biosynthetic studies of families carrying hemoglobin J alpha Mexico: association of alpha-thalassemia with HbJ.

Hemoglobin J Mexico, an alpha chain mutant, was studied in eight unrelated Algerian families. The quantities of the abnormal hemoglobin in 116 subjects are trimodally distributed: 55% in homozygotes, 31% and 38% in heterozygotes. Both hematological data and the alpha/beta chain biosynthetic ratio are normal in heterozygotes with 31% Hb J and in homozygotes. In contrast, the MCV and MCH as well as the alpha/beta biosynthetic ratio are slightly reduced in heterozygotes with 38% Hb J and in their relatives carrying Hb A. The elevated expression of alphaJ chains in heterozygotes with 38% Hb J may be due to an alpha thalassemia gene trans to the alphaJ locus.

Homozygous cases for hemoglobin J Mexico (alpha54 (E3)Gln replaced by Glu) evidence for a duplicated alpha gene with unequal expression.

Hemoglobin J Mexico has been found in five generations of a large Algerian family. Nine subjects have 55% Hb J although their parents, siblings and offspring may have 31%, the usual quantity found in heterozygotes. Those with 55% Hb J are considered to be homozygous for a chromosome carrying both a normal alpha chain locus and a locus for alphaJ. The proportion of the abnormal hemoglobin in all the subjects is in favor of an unequal expression of both loci, the amount of protein synthesis directed by the alpha J gene being greater than that directed by the alpha A. In two heterozygotes a slightly higher proportion of the Hb J (38%) suggests the presence of a single normal alpha chain locus in trans. An associated alpha-thalassemia was excluded by biosynthetic studies in this family.

Structural analysis of the 5' flanking region of the beta-globin gene in African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation in Africa.

Haplotype analysis of the beta-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT)n and (AT)xTy, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. Nevertheless, the haplotype determination does not give any information about the variable segment and does not totally exclude the possibility of recombination leading to different haplotypes linked to the mutation. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT)n and (AT)xTy repeats. We found three additional structures for (AT)xTy correlating with the geographic origin of the patients. Ten other nucleotide positions, 5' and 3' to the (AT)xTy copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5' flanking region of the beta-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

Evolution of the primate beta-globin gene region: nucleotide sequence of the delta-beta-globin intergenic region of gorilla and phylogenetic relationships between African apes and man.

A 6.0-kb DNA fragment from Gorilla gorilla including the 5' part of the beta-globin gene and about 4.5 kb of its upstream flanking region was cloned and sequenced. The sequence was compared to the human, chimpanzee, and macaque delta-beta intergenic region. This analysis reveals four tandemly repeated sequences (RS), at the same location in the four species, showing a variable number of repeats generating both intraspecific (polymorphism) and interspecific variability. These tandem arrays delimit five regions of unique sequence called IG for intergenic. The divergence for these IG sequences is 1.85 +/- 0.22% between human and gorilla, which is not significantly different from the value estimated in the same region between chimpanzee and human (1.62 +/- 0.21%). The CpG and TpA dinucleotides are avoided. CpGs evolve faster than other sequence sites but do not confuse phylogenetic inferences by producing parallel mutations in different lineages. About 75% of CpG doublets have become TpG or CpA since the common ancestor, in agreement with the methylation/deamination pattern. Comparison of this intergenic region gives information on branching order within Hominoidea. Parsimony and distance-based methods when applied to the delta-beta intergenic region provide evidence (although not statistically significant) that human and chimpanzee are more closely related to each other than to gorilla. CpG sites are indeed rich in information by carrying substitutions along the short internal branch. Combining these results with those on the psi eta-delta intergenic region, shows in a statistically significant way that chimpanzee is the closest relative of human.

Nucleotide sequence of the delta-beta-globin intergenic segment in the macaque: structure and evolutionary rates in higher primates.

A 5600-base-pair (bp) fragment including the beta-globin gene and about 4000 bp of its 5' flanking sequence was cloned from the DNA of Macaca cynomolgus (an Old World monkey), and the 5' flanking region was sequenced. Comparison with human, chimpanzee, mouse, rabbit, and Xenopus orthologous sequences reveals a tandemly repeated sequence called RS4 at the same position (about 500 bp 5' from the transcription start of the adult beta-globin gene) in all six species. We suggest that a tandemly repeated sequence has been maintained by functional constraints since the divergence between amphibians and reptiles. Excluding tandemly repeated sequences as well as about 400 nucleotides upstream from the cap site, the average base substitution frequencies among human, chimpanzee, and macaque intergenic sequences were calculated. They appear to be strongly correlated with the delta T50 values measured between the corresponding nuclear DNAs. They are also similar to base substitution frequencies calculated by Chang and Slightom (1984) at the pseudo-eta-globin locus. Thus, exclusion of sequences involved in specific modes of variation might allow the use of intergenic sequences for the accurate calculation of genetic distances. Using a time scale based on the dating of the Atlantic split, we estimate the base substitution rate of primate noncoding DNA to be 1.0 X 10(-9) substitution/site/year.

Nucleotide sequence of the beta-globin genes in gorilla and macaque: the origin of nucleotide polymorphisms in human.

Part of the beta-globin genes of Macaca cynomolgus and Gorilla gorilla has been cloned and sequenced. Ten putatively neutral nucleotide polymorphisms have been described at the beta-globin locus in humans. They are associated in seven combinations, which define seven different haplotypes of the beta-globin gene: four major frameworks--1, 2, 3, and 3--and three minor frameworks, which we term KI1, KA1, and OR1. The nucleotide sequences of these frameworks are compared with those of homologous sequences in chimpanzee, colobus, macaque, and gorilla. This comparison provides strong evidence that framework 2 was the earliest framework in the human lineage. From framework 2, a rooted parsimonious tree for the six other frameworks is constructed. This phylogenetic tree is discussed in terms of the evolution of nucleotide polymorphisms as well as in terms of genetic affinities between human populations. For each position at which there is base difference in comparing human, gorilla, and chimpanzee beta-globin genes, the phyletic lineage where the corresponding substitution occurred has been identified using the maximum parsimony procedure. The data provide evidence that polymorphisms may represent a significant component of differences between closely related species. If so, nucleotide polymorphisms may strongly bias estimates of small evolutionary distances.

[1st cases of alpha-thalassemia in Algeria: 12 cases of hemoglobinosis H]. / Premières observations d'alpha-thalassémie en Algérie: 12 cas d'hémoglobinose H.

These first cases of hemoglobinosis H show that alpha-thalassemia is not a simple curiosity in this part of the Western Mediterranean. They are not localised to a single part of Algeria as the areas from which the patients came were more than 150 km apart. They suggest that the enquiry should be continued by other means to determine the prevalence and pathological incidence.

[Clinical and biological aspects of beta-thalassemia. Apropos of 176 cases]. / Aspects cliniques et biologiques des bêta-thalassémies. A propos de 176 observations.

The study of 176 subjects with beta-thalassemia, associated or not with a hemoglobinopathy, shows great diversity. The hemoglobin C thalassemias are less severe and form a fairly homogeneous group. Sickle cell thalassemia cases have more marked anemia and the disease takes on more varied forms, no doubt because the main mechanism of the anemia, the hyperhemolysis, is influenced by several factors which have a variable effect on the clinical picture. Unassociated thalassemias seem the most polymorphic. Although it seems that in certain foci the beta-thalassemias are fairly stereotyped, this first study shows in Algeria great heterogeneity. All forms are observed both clinically and in the laboratory. Present classifications have not supplied a sufficiently operative model. It is not doubt necessary to await further progress in the laboratory to classify these diseases more precisely.

Mapping the alpha-globin genes in Hb J Mexico carriers.

The organization of the alpha-globin genes was studied by restriction endonuclease mapping, in subjects carrying the alpha variant Hb J Mexico. A subject homozygous for Hb J synthesized both Hb J (about 55%) and Hb A and had two alpha loci per chromosome. His restriction site map was found to be identical to that obtained with a normal DNA, except for a mutant Bgl II site which was observed on the Hb J chromosome proximal to the 5' alpha-locus. We have also mapped the DNA of a compound heterozygote for Hb J and alpha-thalassemia, who synthesizes 38% Hb J and we have found a single alpha gene corresponding to a - alpha 3.7 haplotype on one chromosome and two alpha genes, respectively alpha J and alpha A, on the other.

beta+ -Thalassemia intermedia. Genetic and biochemical study of a family including 3 cases.

3 cases of thalassemia intermedia have been found in the same family. The parents are not consanguineous but both come from the same town of Calabria (Italia). The mother is a heterozygote for beta-thalassemia, as well as the father whose globin chain synthesis is nevertheless balanced, thus suggesting an association with alpha-thalassemia. This hypothesis is confirmed by the fact that one of the offspring shows the typical characteristics of alpha-thalassemia heterozygosity. The 3 subjects with thalassemia intermedia are synthesizing the beta-globin chain in a proportion higher than that expected from the level of Hb A in peripheral blood. In 2 of them, the globin chain biosynthetic ratio measured in the blood reticulocytes is not significantly different from that usually observed in thalassemia major of either the beta o or beta+ type. In the third subject the globin chain synthesis is slightly less unbalanced probably because an alpha-thalassemia is also present. This suggests that factors other than a lesser imbalance in globin chain synthesis are involved in the occurrence of thalassemia intermedia. One of these factors could be a better survival of cells richer in Hb F than in Hb A, since these cells must have a lesser excess of alpha-chains.

Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

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.

[Genetic studies of relationship between Mediterranean and Atlantic populations of loggerhead turtle Caretta caretta with mitochondrial marker]. / Etude génétique des relations entre les populations méditerranéenne et atlantique d'une tortue marine (Caretta caretta) à l'aide d'un marqueur mitochondrial.

The loggerhead turtle Caretta caretta is an endangered species in the Mediterranean. Therefore, the definition of the Mediterranean population, and their relationships to the Atlantic population is of fundamental importance. For this purpose, we have sequenced a portion of the mitochondrial cytochrome b gene to generate genetic markers. Results indicate that the Mediterranean nesting female population is genetically isolated from the Atlantic nesting female population, but loggerhead turtles of Atlantic origin were found in the West Mediterranean basin. This entry of Atlantic loggerheads in the Mediterranean confirms earlier speculations and presents special conservation problems. The Spanish swordfish longline fishery which incidentally captures large numbers of loggerheads in the West Mediterranean basin has therefore an impact on the Atlantic population. These data demonstrate the international nature of marine turtle conservation.

Recent insertion of an Alu sequence in the beta-globin gene cluster of the gorilla.

We present the nucleotide sequence of a new Alu family member that lies between the delta- and beta-globin genes in gorilla DNA. The sequence exhibits 91% similarity with a consensus sequence of the Alu family. It is flanked by a perfect repetition of a 16-nucleotide target sequence and terminates with 24 adenylic residues. As this sequence is absent at this locus in other primate DNAs, its insertion occurred less than 8 million years ago, thus supporting the idea that Alu sequences are still mobile elements in the hominoid genome.