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We evaluated the effects of polymorphic markers within the β globin gene cluster on HbF expression in two groups. These groups were randomly selected from a survey of HbF distribution in a large population study of unrelated healthy Algerian adults (n=827). The first group contained individuals with normal HbF levels (0.1-0.5%) and the second group contained individuals with raised HbF levels (0.8-2.3%). Of the various polymorphic markers analysed, only the -309 Gγ A→G, the -158 Gγ C→T, the Gγ IVS2 TC (TG)[sub 9] AG (TG)[sub 2] (CG)[sub 2] and the -540β (AT)[sub 9] T[sub 5] sequence configurations were significantly associated with increased HbF levels. More than 84% of the subjects with elevated HbF levels carried one or several of these four marker configurations, suggesting that the β giobin gene cluster exerts a significant effect on HbF expression in healthy individuals. [ABSTRACT FROM AUTHOR]

Mutations in the RPS19 gene have been identified in 25% of individuals affected by Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia characterized by an aregenerative anemia and a variety of malformations. More than 60 mutations in the five coding exons of RPS19 have been described to date. We previously reported a mutation (c.-1 + 26G>T) and an insertion at -631 upstream of ATG (c.-147_-146insGCCA) in the noncoding region. Because DBA phenotype is extremely heterogeneous from silent to severe and because haploinsufficiency seems to play a role in this process, it is likely that genetic variations in the noncoding regions affecting translation of RPS19 can modulate the phenotypic expression of DBA. However, to date, very few studies have addressed this question comprehensively. In this study, we performed detailed sequence analysis of the RPS19 gene in 239 patients with DBA and 110 of their relatives. We found that 6.2% of the patients with DBA carried allelic variations upstream of ATG: 3.3% with c.-1 + 26G>T; 2.5% with c.-147_-146insGCCA; and 0.4% with c.-174G>A. Interestingly, the c.-147_-146insGCCA, which has been found in a black American and French Caribbean control population, was not found in 500 Caucasian control chromosomes we studied. However, it was found in association with the same haplotype distribution of four intronic polymorphisms in our patients with DBA. Although a polymorphism, the frequency of this variant in the patients with DBA and its association with the same haplotype raises the possibility that this polymorphism and the other genetic variations in the noncoding region could play a role in DBA pathogenesis.

Human mannose- binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p=0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%,respectively; p=0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV- positive patients.

Human mannose-binding lectin (MBL) plays an important role in innate immunity. MBL deficiency is associated with mutations in the promoter region and in exon 1 of the MBL2 gene. Such deficiency has been correlated with elevated incidence of infections in infancy and in immunocompromised adults. We determined the distribution profile of the MBL2 gene variants in the general population of Benin (West Africa) and in a vulnerable subset of children with sickle cell disease (SCD) (SS homozygotes). Five hundred forty-two healthy individuals (274 newborns, 268 adults) and 128 patients with SCD (35 newborns, 93 children) were screened for the common variant alleles in the MBL2 secretor haplotype region (exon 1 and promoter). The p.G57E variant allele was the most frequent allele compared to p.G54D (27.5% vs. 1.6%, respectively). The p.R52C allele was not found in this population. There was no difference in allele or genotype frequencies between healthy newborns and newborns with SCD. Alleles associated with MBL deficiency were more frequent in adults than in newborns (69.8% vs. 57.3%, respectively; p = 0.002). This enrichment was exclusively due to an elevated proportion of heterozygotes for the p.G57E allele (47.0% vs. 35.3%, respectively; p = 0.004), supporting a potential selective advantage of this genotype. Our results, compared to those reported in other African countries, support the implication of the MBL2 gene in various major infections in Africa, such as meningitis and tuberculosis in HIV-positive patients.

This study identified and characterized a novel delta beta fusion gene in which the delta-globin gene promoter is linked to intact beta-globin coding sequences in a Senegalese family. It results from a 7.4-kb deletion that removes the delta-globin coding sequences, the delta beta intergenic region as well as the beta-globin gene promoter and causes delta(0)beta(+) thalassemia with hemoglobin A expressed at the 11% to 15% range. The phenotype of this naturally occurring delta beta hybrid gene not only clarifies, in an in vivo context, the respective strength of delta- and beta-globin gene promoters, but also emphasizes the importance of beta-globin intragenic sequences in the expression of beta-globin chains. (Blood. 2001;98:1261-1263)

The beta thalassemia alleles in 53 thalassemic Indo-Mauritian patients and their families consisting of 23 homozygous beta-thalassemia, 9 HbE/beta-thalassemia, 18 HbS/beta-thalassemia, 1 HbD/beta-thalassemia, 1 deltabeta/beta-thalassemia and 1 HbH/beta-thalassemia from the island of Mauritius were studied. Characterization by polymerase chain reaction-based reverse dot blot hybridization technique revealed that the IVS1-5 (G-->C) mutation accounted for 74% of the beta thalassemic alleles, while six other mutations occurred at much lower frequencies: HbE codon 26 (G-->A); 10.4%, codon 8/9 (+G); 3.5%, codon 30 (AGG-->ACG) also called IVSI (-1).G-->C; 3.5%, codon 15 (G-->A); 3.5%, codon 41/42 (-CTTT); 2.4% and -28 (A-->G); 2.4%. Association of these mutations to specific beta globin gene sequence framework and haplotype allowed to trace their ancestral link. These data are useful in future molecular screening of the population in view of implementing a thalassemia prevention and control program in Mauritius., (Copyright 2000 Wiley-Liss, Inc.)

We have studied the cellular and molecular responses to long-term hydroxyurea (HU) treatment in 29 severely affected young patients with sickle cell disease (mean age, 10.9 +/- 4.1 years). Patients received HU at 20 mg/kg/d on 4 consecutive days per week initially, with a monthly escalated dose avoiding marrow-toxicity (mean steady-state dose, 34.2 +/- 4.6 mg/kg/d) for 12 to 36 months (mean duration, 22 months). The studied parameters were hemoglobin F (HbF), F reticulocytes (F retics), F cells, the amount of HbF per F cell (F/F cell), polymer tendency at 40% and 70% oxygen saturation, and hemolysis. Initial HbF (Fi) was dispersed (from 0.85% to 13.9%). HbF increased in all patients but 1. HbF at maximal response (Fmax) reached a sustained level varying from a 1.5-fold to a 16-fold Fi after a variable delay (6 to 24 months). Fmax was not related to HU dosage, but triangle upF (Fmax - Fi) was strongly correlated to triangle upMCV (MCVmax - MCVi). HbF increase resulted from the increase of both F cells and F/F cell. In this rather short series, Fi and Fmax were not significantly associated with age, gender, or beta-globin haplotype. Neither Fmax nor triangle upF was related to bone marrow reserve, as measured by baseline reticulocyte or neutrophil counts. However, Fmax was highly dependent on Fi. When patients are individualized into three groups according to Fmax (group 1, Fmax >20% [12 patients]; group 2, 10% < Fmax < 20% [11 patients]; group 3, Fmax <10% [5 patients]), Fi is significantly different between groups, being the highest in group 1. In addition, the best responders (group 1) were significantly different from patients in the two other groups with higher levels of total hemoglobin, decreased bilirubin, and decreased polymer tendency.

Beta thalassemia and Hb Lepore heterozygotes included in this study exhibit fetal hemoglobin levels varying from trace quantities to 14% (1.74 g/dl) of total hemoglobin in the adult. In this work, we have examined the correlation of DNA sequence polymorphisms with the observed HbF level. The analysis of polymorphic markers within the beta globin cluster in 39 individuals heterozygous for beta thalassemia or Hb Lepore confirms the previous findings for homozygous beta thalassemia: the presence of both an (AT)9 T5 sequence configuration at position -540 of the beta globin gene and a (C --> T) variation at -158 of the Ggamma globin gene is associated with elevated expression of HbF. However, at least one defective beta globin gene is required to reveal this association. The best evidence is from the study of individuals heterozygous for Hb Lepore with various levels of HbF. In these individuals it was possible to explore the effect of a single (AT)x Ty motif (the other being absent from the rearranged Lepore chromosome) on HbF expression. The presence of the (AT)9 T5 configuration increases HbF level from a median of 0.515 g/dl observed in (AT)7 T7 subjects, to 1.39 g/dl. We confirm the existence of linkage disequilibrium between the (C --> T) variation at -158 of Ggamma gene and the (TG)13 configuration at the second intervening sequence (IVS-2) of Agamma gene and identify two new polymorphisms in this region: (TG)7 (CG)5 (TG)8 linked to haplotype V and (TG)8 (CG)5 (TG)10 linked to haplotype II. This study suggests that two distinct regions of the beta cluster, whether in cis or in trans to each other, can interact to enhance HbF expression when a beta thalassemic determinant is present in heterozigosity.

The presence of Hb Hope associated with Hb S may represent a pitfall (false positive) in the neonatal detection of sickle cell disease by two of the most widely used analytical methods in screening programmes-isoelectric focusing (IEF) and high performance liquid chromatography (HPLC). This example illustrates the need to improve analytical strategies to avoid unnecessary anxiety and summoning of families often from a cultural background in which testing of the father is difficult to obtain. It is suggested that using two independent HPLC procedures might improve the specificity of the screening strategies. Additionally, simple procedures for detection of the most common mutations of the beta globin gene of DNA extracted from dried blood specimens could be easily developed for the control of abnormal samples. These procedures could be introduced into the analytical strategy.

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.

In Caucasians, the R506Q mutation in exon 10 of the factor V gene (FV Leiden) confers an increased risk of thromboembolism. We have scanned this region of the gene for possible mutations in 450 subjects from populations at risk for sickle cell disease (SCD). The R506Q mutation was absent in subjects from sub-Saharan Africa, whereas its allelic frequency was 2.5% in the West Indies. Only one other substitution with no functional consequences in vitro (R485K) was found (32.4% allelic frequency) in sub-Saharan Africa. Thus, we found no mutations in exon 10 of the FV gene constituting an additional risk factor for thrombosis in SCD in sub-Saharan Africa. This suggests that the putative selective advantage conferred by R506Q does not exist in these populations, unless R485K has functional consequences in vivo. If further suggests that R506Q in American Africans is of Caucasian origin. Our data are the first to document ethnic variations in the frequency of the R485K polymorphism.

Two population groups from Western India with a high prevalence of the beta(S) gene, one tribal (Valsad) and the other nontribal (Nagpur), were studied. The beta(S) gene frequency in both populations was similar (0.22 vs. 0.23), but not the clinical expression of sickle-cell anemia (SS): the sickle homozygotes in the tribal group appeared to have a mild clinical course, whereas the majority in the nontribal group exhibited a more severe clinical phenotype. Both tribal and nontribal SS patients had a similarly high mean hemoglobin (Hb)F expression (18.5% vs. 15.5%) and a high number of F cells (72.3% vs. 66.6%). DNA analysis of the beta-globin gene cluster region revealed that in these two populations, this portion of DNA was identical with and corresponded to the typical Arab-Indian haplotype. Nevertheless, in heterozygotes, the mean beta(S) expression was lower (27.9%) in the tribal as compared to the nontribal group (35.5%). The major epistatic factor distinguishing the milder presentation in tribals vs. a more severe manifestation in nontribals was the very high frequency (0.97) of the alpha-thalassemia gene in the former as compared to the latter (0.24). We conclude that the phenotypic expression of sickle-cell anemia, linked to the Arab-India haplotype and expressing similar levels of HbF and F cells, is not uniformly mild in India and that alpha-thalassemia is a powerful and additional epistatic factor in the Indian subcontinent.

The population of the Dogon, located in Mali, is divided in an endogamic Noble class and two endogamic servant castes (Tanners and Blacksmiths). We find that the polymorphic frequencies of beta c, beta S, and, unexpectedly, a mutation of the delta-chain (delta A'), are geographically (valley vs. plateau) as well as social status dependent.

To elucidate the origin and spread of the sickle cell trait into the Portuguese population, we examined nine polymorphic DNA markers within the beta globin gene cluster defining the haplotype. The population sample included 64 sickle-cell-gene-bearing individuals from defined Portuguese-speaking white, black, and Asian Indian populations. The nature and geographic distribution of the different beta S haplotypes in Portugal suggest that the sickle cell trait has been imported twice: between the eighth and the thirteenth centuries from the Mediterranean basin (in association with the Benin haplotype) and after the fifteenth century from black Africa over an Atlantic route (Senegal and Bantu haplotypes).

During normal human development, a switch is classically observed in the relative expression of the two gamma globin genes, the G gamma/A gamma ratio varying from 70/30 at birth to 40/60 by the end of the first year. An exception to this developmental pattern is linked to the presence of an XmnI restriction site at a position -158 to the Cap site of the G gamma gene. Another exception is observed in individuals homozygous for two easily detectable variations of the A gamma gene: the presence of a threonine residue at codon 75 and a HindIII site within the second intron. A 4-bp deletion has been described around position -225 in some thalassemic patients presenting with these variations. In this study, we find this deletion to be haplotype-linked in a series of 156 individuals of various ethnic origins and presenting with various normal and pathological phenotypes. In sickle cell patients heterozygous for this 4-bp deletion, the relative expression of the A gamma genes on the two chromosomes can be measured by estimating the A gamma T and A gamma I chains, the former always being synthesized at a lower rate. These results suggest a functional role for the deleted sequence.

The sickle cell mutation (beta s) arose as at least three independent events in Africa and once in Asia, being termed the Senegal, Benin, Bantu and Indian types respectively. An investigation in Cameroon was carried out to determine whether the atypical sickle genes observed in the neighboring countries are the result of recombination or the presence of a sickle cell mutation of a different genetic origin. It was conducted on 40 homozygous SS patients followed at the Blood Transfusion Center in the capital city of Yaoundé. On 80 beta s chromosomes, 13 exhibited a novel polymorphic pattern that was observed three times in the homozygous state. This chromosome contains an A gamma T gene. The restriction fragment length polymorphism haplotype is different from all the other beta s chromosomes in both the 5' and 3' regions, but has previously been reported in sporadic cases. The (AT)8(T)5 sequence in the -500 region of the beta gene is specific and different from that of the Senegal, Benin, Bantu or Indian beta s genes. All the carriers of this specific chromosome belong to the Eton ethnic group and originate from the Sanaga river valley. This observation strongly argues for yet another independent origin of the sickle cell mutation in Africa, here referred to as the "Cameroon type". The Benin haplotype and a Benin/Bantu recombinant haplotype have been observed in the other studied populations: Ewondo, Bamiléké, Bassa, Yambassa and Boulou.

The origin of the beta C mutation was studied by characterizing nucleotide sequence polymorphisms on beta C chromosomes of patients from various African countries. In the majority of cases, the beta C mutation was found in linkage disequilibrium with a single chromosomal structure as defined by classical RFLP haplotypes, intergenic nucleotide sequence polymorphisms immediately upstream of the beta-globin gene, and intragenic beta-globin gene polymorphisms (frameworks). In addition, three atypical variant chromosomes carrying the beta C mutation were observed, and are most probably explained either by a meiotic recombination (two cases) or by one nucleotide substitution occurring in an unstable array of tandemly repeated sequences (one case). These data demonstrate the unicentric origin of the beta C mutation in central West Africa, with subsequent mutational modification in a small number of instances. The data also supports gene flow of the beta C chromosome from subsaharan Africa to North Africa.

To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.