Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ only at position 136 reflecting the products of two nonallelic gamma-globin genes. At the time of birth, HbF accounts for approximately 70% of the total Hb. The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a gamma- to beta-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as gamma-thalassemias, duplications, triplications, and even sextuplications of the gamma-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of gamma-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.     

HbF in the adult: could its composition discriminate normal from abnormal foetal globin gene expression?

The relative proportions of the two gamma chain species (G gamma and A gamma) have been reinvestigated in newborns, during the physiological switch from foetal to adult haemoglobin, and in adults with some persistent expression of HbF. In newborns, with about 80% HbF, the G gamma percentage was close to 70% while in adult RBC, with less than 0.5% HbF, the G gamma chain was almost non-detectable and may reflect the completion of the foetal to adult switch. Conversely, in adult patients with HbF above 0.6%, usually accompanying some degree of marrow stimulation, the relative ratio of G gamma varied between 40 and 60%, independently of HbF level. This ratio corresponds to what has been described in the literature as being the adult type of HbF. In all the cases where we found higher levels of G gamma, the results could be explained by the presence of a specific genetic background such as the Senegalese haplotype in sickle cell disease.     

Simplified flow cytometric method for fetal hemoglobin containing red blood cells

Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.     

Molecular characterization of a novel form of (A gamma delta beta)zero-thalassemia deletion with a 3'' breakpoint close to those of HPFH-3 and HPFH-4: insights for a common regulatory mechanism.

We have molecularly characterized a novel (A gamma delta beta)zero-thalassemia associated with increased synthesis of HbF in three members of a German family. The levels of HbF in the peripheral blood red cells of the heterozygotes ranged between 9.9% and 12.5% with a heterocellular distribution in the red cells, as detected by immunofluorescence. The mutation resulted from a deletion starting about 1.5 to 1.9 kb from the 3' end of the G gamma-gene and ending 27 +/- 0.5 kb 3' to the beta-globin gene. Thus, the total deletion is 52 +/- 0.5 kb. Its 5' breakpoint is similar to that of the previously described (A gamma delta beta)zero-thalassemias, while the location of the 3' breakpoint is placed very close to the 3' breakpoints of HPFH-4 and HPFH-3 deletions. The proximity of the 3' breakpoint of the German (A gamma delta beta)zero-thalassemia to those of HPFH-3 and HPFH-4 deletions raises the possibility that a common mechanism, such as the juxtaposition of an enhancer, might underlie the activation of the gamma-globin genes in these three mutants.     

G gamma and A gamma globin genes are identical from -471 of the promoter midway through gamma IVSII in a Benin beta s haplotype associated with elevated fetal hemoglobin.

In seven kindreds in which sickle cell (SS) patients had elevated (greater than 12%) fetal hemoglobin (Hb F), Milner and colleagues reported that a determinant for elevated Hb F and elevated F cells was linked to the beta s gene. Independently, the Senegal (SEN) beta s haplotype has been found in association with elevated Hb F in SS and beta-thalassemia patients. We have used the kindreds of Milner and colleagues to characterize further the association of haplotype and gamma gene DNA sequence variation with Hb F expression. For the largest kindred, Wi, all four SS had high (greater than 14%) Hb F and both SEN and Benin (BEN) haplotypes. Two AS cases carrying SEN had low Hb F and low F cells, while three AS and one CS carrying BEN had elevated Hb F and elevated F cells; only one AS carrying BEN had low Hb F and low F cells. In order to look for genetic alterations that could account for the elevated Hb F of kindred Wi, we sequenced both the G gamma and A gamma genes of the Wi BEN haplotype. The data showed largely identical G gamma and A gamma genes which may have been generated by two gene conversions: the A gamma promoter was like that of G gamma 3' to -471, while the G gamma IVSII was like that of A gamma in its 5' half. In addition, three new mutations were found in gamma IVSII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium

Activated neutrophils increase erythrocyte phosphatidylserine (PS) exposure. PS-exposed sickle red blood cells (SSRBCs) are more adhesive to vascular endothelium than non-PS-exposed cells. An increase in SSRBC fetal hemoglobin (HbF) concentration has been associated with improved rheology and decreased numbers of vasoocclusive episodes. This study examined the effects of HbF, PS-exposed SSRBCs, and chronic hydroxyurea (HU) treatment on activated neutrophil-mediated SSRBC retention/adherence in isolated-perfused rat lungs. Lungs were perfused with erythrocyte suspensions from 1) individuals homozygous for hemoglobin S with 0-7% HbF (SS), 2) with > or =8% HbF (SS + F), and 3) individuals homozygous for hemoglobin S treated with HU therapy for > or =1 yr (SS + HU). Retention of SSRBCs from the SS + HU group was significantly less than that seen in both the SS and SS + F groups. No difference was observed between the SS and SS + F groups. The percentage of HbF and F-cells did not differ between the SS + F and SS + HU groups. At baseline, the proportion of PS-exposed SSRBCs was not different between the SS and SS + HU groups. However, SSRBC treatment with activated neutrophil supernatant caused a twofold increase in PS-exposed SSRBCs in the SS control and no change in the SS + HU group. We conclude that 1) HU attenuates SSRBC retention/adherence in the pulmonary circulation seen in response neutrophil activation, 2) HU stabilizes SSRBC membrane PS, and 3) HU attenuation SSRBC retention/adherence in the pulmonary circulation occurs through a mechanism(s) independent of HbF.     

Comparative spin-label study of the dynamic structure of human hemoglobins A and F and the influence of the antibiotic, chloramphenicol

Hemoglobin A (HbA) and hemoglobin F (HbF) dynamic structures have been studied using spin-label ESR spectra analysis technique, which permits quantitative separation of slow macromolecular rotation (described by rotational correlation time, tau c) and fast anisotropic nitroxide radical motion (described by the 'order parameter', S). The hardly restricted motion of the maleimide spin-label reflects the overall macromolecular rotation and small dynamic structure differences between HbA and HbF were observed (tau c is equal to 26 and 27 ns, respectively). On the other hand, the dynamic equilibrium of the iodoacetamide spin-label demonstrates significant differences between beta- and gamma-chain C-terminus flexibility. Thus, there are different states of alpha,beta and alpha,gamma intersubunit contacts which may be expected to determine the different O2 affinity of HbA and HbF. The antibiotic, chloramphenicol, strongly affects the O2 affinity and the Hill constant of HbF, and also provides detectable changes of gamma-subunit C-terminus flexibility (tau c changes from 20 ns to 27 ns after chloramphenicol treatment of HbF), while the HbA tetramer structure remains almost unaffected. The HbF domain structure rearrangements are accompanied by a decrease of the steric restriction of the spin-label motion (S changes from 0.75 to 0.72).     

K(+) transport in red blood cells from human umbilical cord

The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.     

Transposing sequences between fetal and adult hemoglobins indicates which subunits and regulatory molecule interfaces are functionally related

To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity.     

Induction of foetal haemoglobin synthesis in erythroid progenitor stem cells: mediated by water‐soluble components of Terminalia catappa

Current novel therapeutic agents for the treatment of sickle cell anaemia (SCA) focus on increasing foetal haemoglobin (HbF) levels in SCA patients. Unfortunately, the only approved HbF‐inducing agent, hydroxyurea, has long‐term unpredictable side effects. Studies have shown the potential of plant compounds to modulate HbF synthesis in primary erythroid progenitor stem cells. We isolated a novel HbF‐inducing Terminalia catappa distilled water active fraction (TCDWF) from Terminalia catappa leaves that induced the commitment of erythroid progenitor stem cells to the erythroid lineage and relatively higher HbF synthesis of 9.2‐ and 6.8‐fold increases in both erythropoietin (EPO)‐independent and EPO‐dependent progenitor stem cells respectively. TCDWF was differentially cytotoxic to EPO‐dependent and EPO‐independent erythroid progenitor stem cell cultures as revealed by lactate dehydrogenase release from the cells. TCDWF demonstrated a protective effect on EPO‐dependent and not EPO‐independent progenitor cells. TCDWF induced a modest increase in caspase 3 activity in EPO‐independent erythroid progenitor stem cell cultures compared with a significantly higher (P?0.05) caspase 3 activity in EPO‐dependent ones. The results demonstrate that TCDWF may hold promising HbF‐inducing compounds, which work synergistically, and suggest a dual modulatory effect on erythropoiesis inherent in this active fraction. Copyright © 2014 John Wiley & Sons, Ltd.     

Synergistic effect of two β globin gene cluster mutations leading to the hereditary persistence of fetal hemoglobin (HPFH) phenotype

Co-inheritance of gamma and beta globin gene mutations in a compound heterozygous state is rare but of clinical interest as it provides an important data on understanding the HbF expression. Hematological analysis was carried out (Sysmex KX-21). F-cells were enumerated using flow cytometry. Beta globin gene was analysed by CRDB technique and by DNA sequencing. Gamma globin promoter region was sequenced and expression studies were carried out using real time Taqman assay. We report a family, where two inherited defects of the β globin gene cluster segregate. The proband and her sibling were compound heterozygotes for a novel ^Gγ promoter mutation and the 619 bp deletion a common Indian β thalassemia mutation. Molecular characterization revealed that the father (HbA₂ 5.1%, HbF 5.4%), proband (HbA₂ 3.6%, HbF 31.7%) and her brother (HbA₂ 3.9%, HbF 23.6%) were heterozygous for the 619 bp deletion. The mother (HbA₂ 2.1%, HbF 3.4%) had a normal β globin gene. As both the children showed high HbF levels, the γ globin gene work up was carried out. The ^Gγ-globin gene promoter analysis revealed that the mother and the two children were heterozygous for a 5 bp deletion -ATAAG (-533 to -529) that resides in the GATA binding site. These findings suggest that the 5 bp deletion in the ^Gγ globin promoter has a functional role in silencing the γ-globin gene expression in adults by disrupting GATA-1 binding and the associated repressor complex and results in the up-regulation of gamma globin gene expression. When co-inherited with β -thalassemia trait it leads to a phenotype of HPFH.     

Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease

The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mixtures of 20% and 30% HbF with HbS. We find that the present model for polymerization, including molecular crowding, can accurately predict the rates of such mixtures, by using the single assumption that no significant amount of HbF enters the polymer. The effects of replacing HbS by HbF on the rates of polymer formation are found to be significantly lower than previous measurements appeared to indicate because the impact of the replacement is also highly dependent on the total hemoglobin concentration. This is because the molecular crowding of non-polymerizing HbF offsets substantially the effects of decreasing the concentration of HbS concentration, an effect that increases with concentration. Most strikingly, the demonstrated benefit of hydroxyurea therapy in slowing the kinetics of intracellular polymerization cannot be primarily due to enhanced HbF, but must have some other origin, which could itself represent a promising therapeutic approach.     

Platelet-activating factor (PAF) is the effector of IFN gamma-stimulated invasiveness and motility in a B16 melanoma line

In this study, we investigated whether PAF synthesized by F10-M3 cells (a clone of B16-F10 melanoma line) mediates the increased capacity of these cells to penetrate into Matrigel upon stimulation with IFN gamma. The determination of PAF synthesized by IFN gamma-stimulated tumor cells revealed that 70% of newly synthesized PAF was released into growth media, while the remaining 30% was associated with the cell bodies. An experimental protocol based on the use of WEB 2086, a PAF receptorial antagonist, was designed to explore which of the two fractions of PAF synthesized by IFN gamma-stimulated F10-M3 cells (released into the growth medium or associated with the cell bodies) is essential to their capacity to migrate through Matrigel. We found that the PAF secreted into growth medium is the fraction responsible for the enhanced invasiveness of melanoma cells stimulated with IFN gamma. We also investigated whether motility of melanoma cells is stimulated by IFN gamma, and, if so, whether PAF is involved in this effect. We found that WEB 2086 prevented the remodeling of stress fibers, examined as an index of cell motility, that we observed in F10-M3 cells stimulated with IFN gamma. Furthermore, the observation that PAF receptor is expressed in IFN gamma-stimulated melanoma cells suggests that the invasive phenotype (e.g. migration through a reconstituted basement membrane and motility) promoted by PAF is based on an autocrine mechanism. On the whole, these results might indicate that PAF contributes to the expression of properties typical of an invasive phenotype in tumor cells stimulated with cytokines.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry

The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell on the sIgD+, sIgM+, and I-A+ cells averaged 2 X 10(4) receptors, and no correlation between the levels of expression of Fc gamma R and the B cell markers was evident. The sIgG+ B cells, however, expressed more Fc gamma R (8 X 10(4) receptors/cell) than sIgM+ and sIgD+ B cells. Fc gamma R on splenic macrophages were examined by double labeling spleen cells for Fc gamma R and Mac-1. The Mac-1+ cells (2 to 16% of the spleen cells) were 100% Fc gamma R+ and expressed threefold to fivefold higher numbers of Fc gamma R per cell than the sIgM+ or sIgD+ B cells. The Fc gamma R on T cells were studied on cells double labeled for Fc gamma R and Thy-1, Lyt-1, or Lyt-2. An average of 20% of the T cells expressed Fc gamma R and at least two subsets of Fc gamma R+ T cells were evident: Lyt-2- cells, most of which expressed intermediate (2 X 10(4) Fc gamma R/cell) levels of Fc gamma R, and Lyt-2+ cells, which expressed mainly high (8 X 10(4) Fc gamma R/cell) amounts of Fc gamma R. The levels of expression of Fc gamma R and sIgM increased dramatically in response to infection and were elevated in mice with genetic defects. We conclude that the level of Fc gamma R expression is a characteristic property of subsets of spleen cells from normal and infected mice.     

The distribution of fetal hemoglobin and the types of gamma chain in red cell fractions separated by gradient centrifugation from blood of patients with sickle cell anemia and other hemoglobinopathies

Isopycnic separations of red cells from cord bloods, and from patients with sickle cell anemia, different forms of HPFH, S-beta O-thalassemia, and a beta +-thalassemia homozygosity were made in order to evaluate the distribution of Hb F and the relative levels of G gamma and A gamma chains over the cell fractions. As expected, the cord blood data showed decreased levels of both Hb-F and G gamma chains in the top cell fractions since the beta leads to gamma and high G gamma: A gamma low G gamma: A gamma switches are operative around the time of birth. Complete cell fractionations were made on the blood of three SS patients with low G gamma values (40%) and three SS patients with high G gamma values (60%). The proportion of G gamma chain was constant in all cell fractions, while the Hb-F level was higher in cells with higher densities. The difference in the quantities of the three types of gamma chain in the fetal hemoglobins of two SS patients with an A gamma T heterozygosity, one having a low G gamma value and the other a high G gamma level, can be explained by assuming an alteration in a regulatory mechanism. Considerable variation both in the level of Hb F and in the percentage of G gamma chain was observed in two G gamma A gamma-HPFH heterozygotes with a relatively low G gamma percentage of 30%; an inverse relationship was present between the two parameters. Such a phenomenon was not evident for the G gamma A gamma-HPFH homozygote, and also did not exist in two additional G gamma A gamma-HPFH heterozygotes with an associated alpha-thalassemia-2 heterozygosity who had a similar amount of Hb F but with higher G gamma values of about 50%. The difference in G gamma values between these two categories of G gamma A gamma-HPFH could be due to a higher affinity of the G gamma chains over A gamma chains for a slightly decreased amount of alpha chains as in an alpha-thalassemia-2 heterozygosity. 2 heterozygosity. Although an increased synthesis of Hb-F with G gamma chains was again observed after in vitro incubation of reticulocytes with [35S]methionine none of the isolated cell fractions contained a Hb F with the alpha 2 G gamma 2 composition.     

Negative Epistasis between Sickle and Foetal Haemoglobin Suggests a Reduction in Protection against Malaria

BackgroundHaemoglobin variants, Sickle (HbS) and foetal (HbF) have been associated with malaria protection. This study explores epistatic interactions between HbS and HbF on malaria infection.MethodsThe study was conducted between March 2004 and December 2013 within the sickle cell disease (SCD) programme at Muhimbili National Hospital, Tanzania. SCD status was categorized into HbAA, HbAS and HbSS using hemoglobin electrophoresis and High Performance Liquid Chromatography (HPLC). HbF levels were determined by HPLC. Malaria was diagnosed using rapid diagnostic test and/or blood film. Logistic regression and generalized estimating equations models were used to evaluate associations between SCD status, HbF and malaria.Findings2,049 individuals with age range 0-70 years, HbAA 311(15.2%), HbAS 241(11.8%) and HbSS 1,497(73.1%) were analysed. At enrolment, malaria prevalence was significantly higher in HbAA 13.2% compared to HbAS 1.24% and HbSS 1.34% (p<0.001). Mean HbF was lower in those with malaria compared to those without malaria in HbAA (0.43% vs 0.82%) but was the reverse in HbSS (8.10% vs 5.59%). An increase in HbF was associated with a decrease in risk of malaria OR=0.50 (95%CI: 0.28, 0.90; p=0.021) in HbAA, whereas for HbSS the risk of malaria increased OR=2.94 (1.44, 5.98; p=0.003). A similar pattern was seen during multiple visits; HbAA OR=0.52 (0.34, 0.80; p=0.003) vs HbSS OR=2.01 (1.27, 3.23; p=0.003).ConclusionHigher prevalence of malaria in HbAA compared to HbAS and HbSS confirmed the protective effect of HbS. Lower prevalence of malaria in HbAA with high HbF supports a protective effect of HbF. However, in HbSS, the higher prevalence of malaria with high levels of HbF suggests loss of malaria protection. This is the first epidemiological study to suggest a negative epistasis between HbF and HbS on malaria.     

Polymorphisms near a chromosome 6q QTL area are associated with modulation of fetal hemoglobin levels in sickle cell anemia.

In patients with sickle cell anemia, fetal hemoglobin (HbF) concentrations vary by 2 orders of magnitude. This variance may be a result of heterogeneity in gene regulatory elements; accordingly, we searched for single nucleotide polymorphisms (SNPs) that might identify this variation. More than 180 SNPs were studied in 38 genes in 280 sickle cell anemia patients. The strongest association with HbF was found with SNPs near a QTL previously localized on chromosome 6q22.3-q23.2. Initially, two SNPs were identified in intergenic portions of this QTL and were associated with about a 20% difference in percent HbF. Subsequently, we genotyped 44 additional SNPs in the genomic region between 136.1 Mb and 137.5 Mb on chromosome 6q. Twelve SNPs, associated with a 20%-30% difference in HbF concentrations, were located in the introns of four genes, PDE7B, MAP7, MAP3K5 and PEX7. In K562 cells, the p38-MAPK pathway has been associated with the activation of gamma-globin gene expression by histone deacetylase inhibitors. Haplotypes C-T-T-T in MAP7 and T-C-C in PEX7 were significantly associated with increases in concentration of HbF, both showing strong dominance. Genetic elements abutting the 6q22.3-q23.2 QTL, may harbor trans-acting elements that help modulate baseline HbF level in sickle cell anemia.