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Patients who are homozygous for the sickle hemoglobin mutation can present with remarkably different clinical courses, varying from death in childhood, to recurrent painful vasoocclusive crises and multiple organ damage in adults, to being relatively well even until old age. Increasing numbers of genetic loci have now been identified that can modulate sickle cell disease phenotype, from nucleotide motifs within the beta-globin gene cluster, to genes located on different chromosomes. With recent success of the human genome project, it is anticipated that many more genetic modifiers of sickle cell disease will be discovered that can lead to the development of more effective therapeutic approaches. The multigenic origin of the variable phenotype in sickle cell disease will serve as a paradigm for the study of variation in phenotypes of all single gene disorders in man.

Homozygous beta thalassemia affects thousands of people around the world. Current management of this condition includes regular transfusion of red cells, which leads to transfusional iron overload requiring chelation therapy: increasing hemoglobin levels while decreasing or eliminating iron overload is therefore a major therapeutic goal in the treatment of thalassemia. Bone marrow transplantation may achieve this goal, but it is not an option for most patients. This study reports on efforts to increase gamma-globin transcription and HbF production using sodium phenylbutyrate (SPB) and hydroxyurea (HU). It was found that 36% (4/11) of all patients or 50% (4/8) of non-transfused patients responded to SPB (increase in Hb levels of 1 g/dL). A positive correlation between baseline serum erythropoietin level and likelihood of response to SPB was observed. Since HU may also increase HbF production, evaluation of combination therapy with these drugs is underway and preliminary results are reported.

Five factors have been hypothesized to influence the 20-fold variation in fetal haemoglobin (Hb F) levels in sickle cell anaemia (SS): age sex, alpha-globin gene number, beta-globin haplotype, and the X-linked F-cell production locus (FCP) that regulates the production of Hb F containing erythrocytes (F cells). We analysed the association of these factors with Hb F levels in 112 SS patients living in France who are homozygous for the three common African beta-globin haplotypes (Benin, Bantu or Central African Republic and Senegal). We found that: (1) FCP accounts for about 40% of the overall variation in Hb F levels, (2) when the FCP influence is removed, beta-globin haplotype is associated with 14% of the remaining Hb F variation, and (3) the other factors have little influence. Comparison with our previous study of SS individuals in Jamaica leads to the following conclusions: (1) the X-linked FCP locus is a major determinant of Hb F levels in SS disease, (2) factors linked to the beta-globin haplotype have only a small effect on the variation in Hb F levels, in either the homozygous or heterozygous state, and (3)approximately half of the variation in Hb F levels still remains to be explained.

Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P < .05). Multiple regression analysis, including all variables, showed that the FCP locus is the strongest predictor, accounting for 40% of Hb F variation. beta-Globin haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.

Patients with sickle cell anemia were treated with daily doses of hydroxyurea, to assess pharmacokinetics, toxicity, and increase in fetal hemoglobin (Hb) production in response to the drug. Plasma hydroxyurea clearances were not a useful guide to maximum tolerated doses of the drug. The mean daily single oral dose that could be maintained for at least 16 weeks was 21 mg/kg (range, 10 to 35 mg/kg). Among 32 patients, last HbF levels were 1.9% to 26.3% (mean, 14.9%) with increases in HbF over initial values of 1.4% to 20.2% (mean, 11.2%). The most significant predictors of last HbF were last plasma hydroxyurea level, initial white blood count and initial HbF concentration. Last HbF was not related to beta globin haplotype or alpha globin gene number. No serious toxicity was encountered. Clinically significant bone marrow depression was avoided, and chromosome abnormalities after 2 years of treatment were no greater than those observed before treatment. The period of observation has been too short to evaluate the risk of carcinogenesis. Patient's red cells developed striking macrocytosis. Median red cell Hb concentrations did not change. Hb concentrations increased, on average 1.2 g/dL, but serum erythropoietin levels increased. Patients' body weights increased, and some returned to work or school, but no conclusions regarding therapeutic efficacy could be drawn from this uncontrolled open-label study.

Single nucleotide substitutions in the promoter regions of the A gamma- and G gamma-globin genes have been associated with increased fetal hemoglobin (HbF) production. We wished to determine whether these or other unrecognized substitutions in the gamma promoter regions are responsible for the 20-fold variation in HbF production in sickle cell patients or normal adults. From a random sampling of 250 sickle cell (SS) patients and 125 normal adults, 17 individuals representing the highest and lowest HbF producers were selected for study. All three common restriction fragment length polymorphism beta-globin region haplotypes (Benin, Central African Republic, and Senegal) were found in both the highest and lowest HbF producers with SS disease. Using the polymerase chain reaction amplification and direct sequencing of the amplified DNA product, we examined the promoter regions of both the A gamma and G gamma genes from -350 bp to +50 bp of the CAP site. No mutations were found in either gamma gene promoter region. We conclude that nucleotide substitutions in the promoter regions (-350 to +50 bp) of both gamma genes are not responsible for the marked variation in HbF production among SS or normal individuals.

We have developed methodology that allows comparison of the mean corpuscular hemoglobin (MCH) of fetal hemoglobin (HbF)-containing red cells (F cells) with the MCH of non-F cells from the same individual. To do this, suspensions of peripheral blood erythrocytes and their internal contents are fixed with an imidodiester, dimethyl-3,3'-dithiobispropionimidate dihydrochloride (DTBP). Thereafter fixed cells are made permeable to antisera by treatment with Triton X-100 and isopropanol, reacted with a mouse monoclonal antibody (MoAb) against HbF, and then with fluorescein-conjugated antimouse IgG. No appreciable hemoglobin is lost during such manipulation. Red cells from a diversity of subjects were thus treated and examined microscopically, first by transmitted light and then by epifluorescence. A direct correlation between Coulter-derived MCH and mean absorbance of 415 nm transmitted light was found for 100 unfixed (r = 0.96) and for 100 antibody-treated fixed-permeabilized red cells (r = 0.99) among individuals selected so as to provide a range of Coulter MCH values between 20 and 35. Comparisons of microscopically derived MCH of F cells and non-F cells were statistically nondistinguishable (P greater than 0.05) in all subjects. Such comparisons included normal individuals (less than 1% F cells), SS patients (7% to 48% F cells), subjects with congenital anemia (22% to 65% F cells), individuals with heterocellular hereditary persistence of HbF (HPFH) (12% to 21% F cells), and heterozygotes for beta + thalassemia (11% to 31% F cells). We conclude that gamma- and beta-globin production within F cells is regulated in a reciprocal fashion both among normal individuals and among individuals with elevated HbF production.

In order to investigate the origin(s) of the mutation(s) leading to the beta S-globin gene in North American populations of African ancestry, we analyzed DNA polymorphisms in the beta-globin gene cluster in a large number of both beta A- and beta S-globin gene-bearing chromosomes in U.S. and Jamaican Blacks. We found 16 different haplotypes of polymorphic sites associated with 170 beta S-globin gene-bearing chromosomes. The three most common beta S haplotypes, which account for 151/170 of the beta S-globin gene-bearing chromosomes, are only rarely seen in the chromosomes bearing the beta A-globin gene in these populations (6/47). Two observations suggest multiple origins or interallelic gene conversion, or both, of the beta S mutation. First, the mutation is present in all three beta-globin gene frameworks. Second, the beta S haplotypes can be divided into four groups, each of which cannot be derived from any other by less than two crossing-over events. In summary, our observation of the beta S mutation on 16 different haplotypes in African populations can be best explained by (i) a number of simple recombination events 5' to the beta-globin gene and (ii) up to four independent mutations and/or interallelic gene conversions.

5-Azacytidine, given to one sickle cell patient as 9 doses of 30 mg/m2 each at intervals of 8 hr, rapidly increased the number of red cells containing HbF (F cells), decreased the amount of HbS in F cells, and decreased the MCHC of F cells. Although a significant rise in the peripheral blood hemoglobin from 8 gm/dl to 10-12 gm/dl was obtained, painful vaso-occlusive crises continued to occur. Therapy was associated with no marrow toxicity, although erythroblastosis appeared after each course of therapy. Increased HbF production was associated with demethylation of two restriction enzyme sites 5' to the two gamma-globin genes. Five other restriction sites around the gamma-globin genes were unchanged. 5-Azacytidine is considered potentially mutagenic and carcinogenic in man and, therefore, further experimental treatment should be limited only to severely affected sickle cell patients.

Fetal hemoglobin (HbF) levels vary widely among individuals with sickle cell anemia (SS). Previous studies have suggested that HbF levels in SS individuals with alpha-thalassemia (two or three functional alpha-globin genes) are lower than HbF levels in SS individuals with four normal alpha-globin genes. Using immunocytochemical techniques, we studied F cell production as measured by % F reticulocytes, the amount of HbF per F cell, and the preferential survival of F cells versus non-F cells in 51 subjects with four alpha genes, 32 subjects with three alpha genes, and 18 subjects with two alpha genes. Comparison between alpha-globin gene groups was performed for the total sample as well as for a subset of 82 individuals who had replicate samples and a further subset of 39 age-matched individuals. %HbF levels were 6.8, 4.9, and 4.5 percent for the total four-, three-, and two-alpha-globin-gene groups, respectively. The percentage of F reticulocytes, percentage HbF per F cell, and the enrichment ratio (% F cell/% F reticulocytes) did not change significantly with alpha-globin gene number. Moreover, no correlation existed between alpha-globin gene number and the absolute number of F cells in any group studied. However, there was a strong inverse correlation (r = -0.407, P = .0001) between non-F cell levels (1.7 +/- 2, 2.2 +/- 5, 3.0 +/- 1.0 X 10(12)/L) and decreasing alpha-globin gene number. These data suggest that falling HbF levels among SS individuals with lessened numbers of alpha-globin genes reflect prolonged survival of non-F cells and are not due to intrinsic differences in F cell production or in the amount of HbF per F cell. The improved survival of non-F cells in SS alpha-thalassemia is presumed to be due to the lower MCHC observed in such individuals.