Effect of beta-globin gene cluster haplotype on the hematological and clinical features of sickle cell anemia

In 113 black American adults with sickle cell anemia (HbSS), we examined nine polymorphic restriction sites, including the Xmnl site 5' to the G gamma gene, to see whether haplotype is related to the level of HbF and the proportion of G gamma chains or if it influences the hematological and clinical features of the disease. Seventy-five percent of the patients were homozygous or heterozygous for the Benin (no. 19) or Central African Republic (Bantu, no. 20) haplotypes; 13.3% were homozygous or heterozygous for the Senegal (no. 3) haplotype, while 11.5% had other genotypes. Of the subjects, 14.2% were either homozygous or heterozygous for the Xmnl restriction site 5' to the G gamma gene. We found no effect of haplotype on HbF levels. The level of G gamma chains was 60.5% +/- 17.0% in individuals heterozygous or homozygous for haplotype no. 3 and was 46.9% +/- 11.6% in individuals with other haplotypes. Subjects with the Xmnl site 5' to the G gamma gene had G gamma globin levels of 59.5% +/- 16.7% while those lacking that site had an average of 47.2% +/- 12.1%. There were no significant differences among these groups in hemoglobin concentration, packed cell volume, mean cell volume, or clinical indicators of vaso-occlusive severity, including crises, hospitalizations per year, aseptic bone necrosis, acute chest syndrome, or leg ulcers. While the presence of haplotype 3 and the 5' G gamma Xmnl site were associated with increased G gamma chains, there was no effect on HbF level or other hematological and clinical features that might reflect disease severity. It is likely that determinants unrelated to haplotype, linked or unlinked to the beta-globin gene cluster, are the major effectors of differences in the levels of HbF in American patients with sickle cell anemia.     

The presence of alpha-thalassaemia trait blunts the response to hydroxycarbamide in patients with sickle cell disease

Hydroxycarbamide (HC), although a key drug therapy in sickle cell disease (SCD), does not result in a clinical response in all patients. Increases in fetal haemoglobin (HbF) and mean corpuscular volume of erythrocytes are standard clinical measures of HC efficacy in SCD. Genetic studies have determined that the majority of HbF regulation occurs outside the beta-globin locus. Approximately 30% of SCD patients have co-inherited alpha-thalassaemia resulting in hypochromic and microcytic erythrocytes. We provide data from 30 SCD patients (10 with alpha-thalassaemia) demonstrating that co-existing alpha-thalassaemia significantly affects several standard measures of HC efficacy in SCD.     

Oxidative stress status,clinical outcome,and β‐globin gene cluster haplotypes in pediatric patients with sickle cell disease

Objective: To correlate the clinical and hematological features of β‐globin gene haplotypes with the oxidative stress status in pediatric patients with sickle cell disease (SCD). Methods: A total of 95 patients with SCD and 40 healthy children were studied. The β‐globin cluster, plasma lipid peroxidation (LPO) and plasma nitrite plus nitrate (NOx), and erythrocyte content of glutathione (GSH) and glutathione disulfide (GSSG), and glutathione peroxidase (GPx), reductase (GRd), and superoxide dismutase (SOD) activities were measured. Results: Plasma LPO (P < 0.001) and NOx (P < 0.05) were significantly higher in patients than in controls. In erythrocytes of patients with SCD, the activities of GRd (P < 0.001) and SOD (P < 0.05) were lower, and the GSSG/GSH ratio (P < 0.001) and GPx activity (P < 0.001) were higher than in controls. High LPO levels and low SOD plus GRd activities were associated with increased severity of clinical manifestations, which correspond mainly to patients with Bantu and Benin haplotypes. LPO levels were reduced in patients with high fetal hemoglobin (HbF) levels, whereas the NOx levels and GRd activity tended to increase in this group. Conclusion: Our results detected an important oxidative stress in patients with SCD and suggest that at least three redox markers, i.e., LPO, GRd, and SOD, were related with their clinical outcomes. Moreover, a relationship between high HbF and low LPO, and high HbF and high GRd activity and NOx levels were found.     

DNA hypomethylation therapy for hemoglobin disorders: molecular mechanisms and clinical applications

Reactivation of fetal hemoglobin (HbF) expression is an important therapeutic option in patients with hemoglobin disorders. In sickle cell disease (SCD), an increase in HbF would interfere with the polymerization of sickle hemoglobin while in beta-thalassemia, an increase in gamma-globin chain synthesis would decrease non-alpha:alpha chain imbalance. Hydroxyurea, an inducer of HbF, is the only currently approved agent for the treatment of patients with moderate and/or severe SCD. However, about one third of patients with SCD do not respond to HU, and in beta-thalassemia, the clinical response is unimpressive. The last decade has seen a renewed interest in the use of inhibitors of DNA methylation in the treatment of patients with hemoglobin disorders. In this review, we discuss the role of DNA methylation in gamma-globin gene regulation, describe clinical trials with agents that hypomethylate DNA and speculate about the future role of DNA hypomethylation therapy in patients with SCD and beta-thalassemia.     

Fetal hemoglobin in sickle cell anemia: The Arab‐Indian haplotype and new therapeutic agents

Fetal hemoglobin (HbF) has well‐known tempering effects on the symptoms of sickle cell disease and its levels vary among patients with different haplotypes of the sickle hemoglobin gene. Compared with sickle cell anemia haplotypes found in patients of African descent, HbF levels in Saudi and Indian patients with the Arab‐Indian (AI) haplotype exceed that in any other haplotype by nearly twofold. Genetic association studies have identified some loci associated with high HbF in the AI haplotype but these observations require functional confirmation. Saudi patients with the Benin haplotype have HbF levels almost twice as high as African patients with this haplotype but this difference is unexplained. Hydroxyurea is still the only FDA approved drug for HbF induction in sickle cell disease. While most patients treated with hydroxyurea have an increase in HbF and some clinical improvement, 10 to 20% of adults show little response to this agent. We review the genetic basis of HbF regulation focusing on sickle cell anemia in Saudi Arabia and discuss new drugs that can induce increased levels of HbF.     

Beta-thalassaemia intermedia in Lebanon

Approximately one third of thalassaemia patients on record in Lebanon have thalassaemia intermedia. We have analysed three factors in a panel of 73 patients with this less severe form of the disease in our population: mild beta-globin gene mutations, deletions in the alpha-globin gene and the presence of a polymorphism for the enzyme Xmn I in the Ggamma-promoter region. The results show that the most important contributing factor is the beta-genotype: 68% of patients have a mild beta+ mutation (IVSI-6, cd29, -88 or -87), while 26% of patients are positive for the Xmn I polymorphism associated with increased production of HbF, which showed strong linkage to particular mutations (IVSII-1, cd8 and cd30). However, the genotype phenotype correlation is difficult, because many patients were initially misdiagnosed as thalassaemia major and were started early on regular blood transfusions, which was stopped later on. This illustrates well the importance of an early accurate diagnosis of thalassaemia intermedia for appropriate clinical management.     

The role of heterocellular hereditary persistence of fetal haemoglobin in beta(0)-thalassaemia intermedia

Beta(0)-thalassaemia intermedia (beta(0)-TI) describes patients who lack beta-globin synthesis yet manifest a non-transfusion-dependent form of beta-thalassaemia. Co-inheritance of alpha-thalassaemia, certain variants of the beta-like globin gene cluster and elevated fetal haemoglobin (HbF) production are all associated with beta(0)-TI. However, the mild phenotypes of many beta(0)-TI patients are unexplained. Genetically determined HbF levels in beta-thalassaemia are difficult to assess because erythrocytes containing HbF (F cells) preferentially survive over erythrocytes without HbF. To evaluate the importance of genetically elevated HbF in beta-thalassaemia, F-cell levels of 19 TI patients' relatives were compared with relatives of transfusion-dependent beta-thalassaemia major patients and those of beta-globin genotype-matched controls. The beta-globin and alpha-globin genotypes, as well as their Ggamma promoter were also examined. Using this approach, in all but one patient the mild phenotype was attributable to either alpha-globin genotype, gamma-globin promoter polymorphism or inherited elevated F-cell levels. The findings of this study establish the F-cell levels required to modify the degree of disease severity significantly and demonstrate that F-cell level is a crucial parameter in the understanding of phenotypic variation in beta-thalassaemia.     

Relationship between the clinical manifestations of sickle cell disease and the expression of adhesion molecules on white blood cells

BACKGROUND: The severity of sickle cell disease (SCD) increases with leukocyte count. The biological basis could be that leukocyte adherence to vascular endothelium mediated by adhesion molecules (AMs) facilitates vaso-occlusion, the basic pathological process in SCD. OBJECTIVE: To find out if there is a relationship between expression of AMs by leukocytes and the clinical manifestations of SCD. METHODS: Flow cytometry was used to study the relationship between leukocyte AM expression and disease manifestations in 100 patients with homozygous (HbSS) sickle cell disease and 34 genotype HbAA controls. The effect of hydroxyurea therapy on AM expression was also examined. We excluded HbSS patients with any other disease, pregnancy in the previous 3 months, or Haemogloben F (HbF) > or = 10%. RESULTS: Patients with complications of SCD showed high expression of alphaMbeta integrin by the neutrophils; and l-selectin by lymphocytes and neutrophils (P < 0.03). CD18 was highly expressed by neutrophils in patients with sickle nephropathy (P = 0.018), and l-selectin by lymphocytes in those with stroke (P = 0.03). Monocyte l-selectin increased in sickle cell crisis relative to steady state (P = 0.04). Expression of alphaLbeta2 integrin by neutrophils, monocytes, and lymphocytes decreased within a month of hydroxyurea therapy (P < 0.05), with symptomatic improvement in the patients and no more than 3.3% rise in HbF level. CONCLUSIONS: The findings suggest that in SCD (1): High steady-state expression of alphaMbeta2 integrin and l-selectin by leukocytes predisposes to severe manifestations. (2) Increased leukocyte AM expression above steady-state levels could be important in the genesis of crisis. (3) The early symptomatic improvement that follows hydroxyurea therapy is mediated via mechanisms independent of increased HbF, and may involve reduced AM expression in leukocytes. (4) Other treatment modalities that reduce leukocyte AM expression might also confer clinical benefit.     

DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease

Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620–1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of ^Gγ-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 × 10⁻⁴²). Together, common SNPs at the BCL11A, HBS1L-MYB, and β-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.     

Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease

Fetal hemoglobin (HbF) level and the HbF responses to hydroxyurea (HU) vary among patients with sickle cell disease and are, at least in part, genetically regulated. We hypothesized that siblings with sickle cell disease are likely to share the same parental beta-like globin gene clusters with their cis-acting regulatory sequences and therefore, if regulation of this response is linked to the beta-globin gene cluster, might have concordant HbF responses to HU. Accordingly, we studied 26 families (30 sib pairings), 20 with sickle cell anemia (three families had three siblings) and 6 families with HbS-beta-thalassemia (one family had three siblings, and one family consisted of monozygotic twins), to see if siblings with sickle cell disease had discordant or concordant changes in HbF during HU treatment. Intraclass correlation coefficients (r) showed a high, positive correlation between sibs for HbF levels before and during HU treatment and a concordant change in HbF response from baseline to treatment-associated levels. Changes in mean corpuscular volume (MCV) paralleled HbF levels, while the expected correlations between treatment-associated fall in leukocyte count and increase in MCV were also present. Our results provide additional evidence that some elements that regulate HbF expression are linked to the beta-globin gene cluster.     

Effect of a group of genetic markers around the 5′ regulatory regions of the β globin gene cluster linked to high HbF on the clinical severity of β thalassemia

The clinical and hematological course of β thalassemia intermedia is influenced by a number of genetic factors which play a role in increasing fetal haemoglobin levels. Several polymorphisms located in the promoters of β and γ globin gene are involved in influencing the disease severity. Our objective was to study the effect of cis-DNA haplotypes, motifs, or polymorphisms (Pre G γ globin gene haplotypes, Aγ–δ intergenic region haplotypes XmnI and (AT)_x(T)_y polymorphisms, β-LCR HS2 and HS3 site motifs) that may contribute to higher HbF levels and a milder clinical course. We found that a combination of T haplotype of the Aγ–δ intergenic region, TAG Pre-Gγ haplotype, presence of the XmnI polymorphism along with the (AT)₉(T)₅ motif constitutes a topography that co-relates with raised HbF levels which may contribute in ameliorating the disease severity.     

MIR29B mediates epigenetic mechanisms of HBG gene activation

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.     

Fetal hemoglobin in sickle cell anemia

Fetal hemoglobin (HbF) is the major genetic modulator of the hematologic and clinical features of sickle cell disease, an effect mediated by its exclusion from the sickle hemoglobin polymer. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable. Some patients with sickle cell disease have exceptionally high levels of HbF that are associated with the Senegal and Saudi-Indian haplotype of the HBB-like gene cluster; some patients with different haplotypes can have similarly high HbF. In these patients, high HbF is associated with generally milder but not asymptomatic disease. Studying these persons might provide additional insights into HbF gene regulation. HbF appears to benefit some complications of disease more than others. This might be related to the premature destruction of erythrocytes that do not contain HbF, even though the total HbF concentration is high. Recent insights into HbF regulation have spurred new efforts to induce high HbF levels in sickle cell disease beyond those achievable with the current limited repertory of HbF inducers.     

Fetal hemoglobin in sickle cell anemia: Bayesian modeling of genetic associations

We genotyped single nucleotide polymorphisms (SNPs) in: (1) the beta-globin gene-like cluster, (2) quantitative trait loci (QTL) previously associated with fetal hemoglobin (HbF) concentration on chromosomes 6q, 8q, and Xp, and (3) candidate genes that could effect HbF levels, in sickle cell anemia subjects. HbF concentration was modeled as a continuous variable with values in a finite interval using a novel Bayesian approach. We first tested the associations of SNPs with HbF in a group of 1,518 adults and children (CSSCD study), and validated the results in a second independent group of 211 adults (MSH study). In subjects aged >or=24 years, 5 SNPs in TOX (8q12.1), 2 SNPs in the beta-globin gene-like cluster, 2 SNPs in the Xp QTL, and 1 SNP in chromosome 15q22 were associated with HbF in the CSSCD and also validated in the MSH. Four other SNPs in 15q22 were associated with HbF only in the larger CSSCD data. When patients aged <24 years in the CSSCD were examined, additional genes, including 4 with roles in nitric oxide metabolism, were associated with HbF level. These studies confirm prior analyses using traditional analytical approaches showing associations of SNPs in TOX, GPM6B, and the beta-globin gene-like cluster with HbF levels. We also identified an additional candidate regulatory region in chromosome 15q22 that is associated with HbF level. By stratifying patients by age, our results also suggest that different genes might modulate the rate of decline of HbF and the final level of HbF levels in sickle cell anemia.     

Variation in hemoglobin F production among normal and sickle cell adults is not related to nucleotide substitutions in the gamma promoter regions.

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.     

Rearrangements of the beta-globin gene cluster in apparently typical betaS haplotypes

BACKGROUND AND OBJECTIVES: The majority of the chromosomes with the betaS gene have one of the five common haplotypes, designated as Benin, Bantu, Senegal, Cameroon, and Arab-Indian haplotypes. However, 5-10% of the chromosomes have less common haplotypes, usually referred to as atypical haplotypes. We have demonstrated that most atypical haplotypes are generated by recombinations. The present study was carried out in order to explore whether recombination also occurs in chromosomes with the common (or typical) haplotypes. DESIGN AND METHODS: We screened the HS-2 region of the beta-globin gene locus control region (LCR) in 244 sickle cell patients who had typical restriction fragment length polymorphism (RFLP)-defined haplotypes of the betaS-gene cluster. For 14 cases in which the expected and the observed LCR repeat-sequence sizes were discrepant, the analysis was extended to other unexplored polymorphic markers of the bS-globin gene cluster, i.e.: pre-Ggamma framework, pre-Ggamma 6-bp deletion, HS-2 LCR (AT)xR(AT)y and pre-beta(AT)xTy repeats, and the intragenic beta-globin gene framework. RESULTS: In all 14 cases (15 chromosomes) in which the LCR repeat-sequence sizes were discrepant, a recombination involving a typical 3' segment of the betaS globin gene cluster was demonstrated. In most of the cases, the recombination site was located between the beta-globin gene and the betaLCR. Nine cases involving recombination were detected among 156 Brazilian HbS homozygotes and five among 88 African patients homozygotes for the Benin haplotype. INTERPRETATION AND CONCLUSIONS. Thus, 3.1% of apparently typical haplotypes linked to the sickle cell gene involve recombinations similar to those that generate the atypical haplotypes, a finding that reinforces the picture of the beta-globin gene cluster as highly dynamic.     

Heritability of fetal hemoglobin,white cell count,and other clinical traits from a sickle cell disease family cohort

Sickle cell disease (SCD) is the most common monogenic disorder in the world. Notably, there is extensive clinical heterogeneity in SCD that cannot be fully accounted for by known factors, and in particular, the extent to which the phenotypic diversity of SCD can be explained by genetic variation has not been reliably quantified. Here, in a family-based cohort of 449 patients with SCD and 755 relatives, we first show that 5 known modifiers affect 11 adverse outcomes in SCD to varying degrees. We then utilize a restricted maximum likelihood procedure to estimate the heritability of 20 hematologic traits, including fetal hemoglobin (HbF) and white blood cell count (WBC), in the clinically relevant context of inheritance from healthy carriers to SCD patients. We report novel estimations of heritability for HbF at 31.6% (±5.4%) and WBC at 41.2% (±6.8%) in our cohort. Finally, we demonstrate shared genetic bases between HbF, WBC, and other hematologic traits, but surprisingly little overlap between HbF and WBC themselves. In total, our analyses show that HbF and WBC have significant heritable components among individuals with SCD and their relatives, demonstrating the value of using family-based studies to better understand modifiers of SCD.     

Discovering the genetics underlying foetal haemoglobin production in adults

Sickle cell disease (SCD) and β thalassaemia, caused by lesions that affect the HBB (β globin gene), form the most common human genetic disorders world-wide, and represent a major public health problem. Inter-individual variation in foetal haemoglobin (HbF) expression is a known and heritable disease modifier; high HbF levels are correlated with reduced morbidity and mortality in both diseases. This review traces our progress in the understanding of the persistence of HbF in adults as a quantitative trait and the genetic approaches used in teasing out the loci contributing to its variability in normal populations and in patients with haemoglobinopathies. Three major loci – Xmn1- HBG2 single nucleotide polymorphism, HBS1L-MYB intergenic region on chromosome 6q, and BCL11A – contribute 20–50% of the trait variance in patients with sickle cell anaemia and healthy European Caucasians. It is likely that the remaining trait variance is due to numerous other loci, many contributing modest effects. Identification of the three major loci has not yet been translated into new therapeutic approaches for HbF reactivation but an immediate application would be an improved prediction of one's ability to produce HbF, which in turn, may improve prediction of disease severity.     

Sickle cell disease in Saudi Arabia: the phenotype in adults with the Arab‐Indian haplotype is not benign

Sickle cell disease (SCD) in Saudi patients from the Eastern Province is associated with the Arab‐Indian (AI) HBB (β‐globin gene) haplotype. The phenotype of AI SCD in children was described as benign and was attributed to their high fetal haemoglobin (HbF). We conducted a hospital‐based study to assess the pattern of SCD complications in adults. A total of 104 patients with average age of 27 years were enrolled. Ninety‐six per cent of these patients reported history of painful crisis; 47% had at least one episode of acute chest syndrome, however, only 15% had two or more episodes; symptomatic osteonecrosis was reported in 18%; priapism in 17%; overt stroke in 6%; none had leg ulcers. The majority of patients had persistent splenomegaly and 66% had gallstones. Half of the patients co‐inherited α‐thalassaemia and about one‐third had glucose‐6‐phosphate dehydrogenase deficiency. Higher HbF correlated with higher rate of splenic sequestration but not with other phenotypes. The phenotype of adult patients with AI SCD is not benign despite their relatively high HbF level. This is probably due to the continued decline in HbF level in adults and the heterocellular and variable distribution of HbF amongst F‐cells.