BCL11A enhancer haplotypes and fetal hemoglobin in sickle cell anemia.

BACKGROUND: Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. METHODS AND RESULTS: Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%. CONCLUSIONS: Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.

Quantitative MRI analysis of craniofacial bone marrow in patients with sickle cell disease.

BACKGROUND AND PURPOSE: Assessment of bone marrow is most commonly performed qualitatively in the spine or other large long bones. The craniofacial bones are less ideal for bone marrow analysis because of the relatively small bone marrow volume. Because patients with SCD often undergo repeated brain imaging to evaluate for cerebral vaso-occlusive disease, quantitative assessment of craniofacial bone marrow is a reasonable possibility in these patients. The purpose of this study was to investigate specific sickle cell disease changes in craniofacial bone marrow quantitatively by analyzing T1, T2, and secular-T2 relaxation times and volume with the use of quantitative MRI. MATERIALS AND METHODS: Fourteen patients with SCD and 17 control subjects were imaged with the mixed TSE pulse sequence at 1.5T. The craniofacial bones were manually segmented by using 3D Slicer to generate bone marrow volumes and to provide T1, T2, and secular-T2 relaxation times. RESULTS: All subjects exhibited a bimodal T1 histogram. In the SCD group, there was a decrease in amplitude in the first T1 peak and an increase in amplitude in the second T1 peak. The first T1 peak showed a significant increase in relaxation time compared with control subjects (P < .0001), whereas there was no significant difference in the second T1 peak. T2 and secular-T2 relaxation times were significantly shorter in the SCD group (T2, P < .0001; secular-T2, P < .0001). Increasing numbers of blood transfusions resulted in a decrease in T2 and secular-T2 times. Patients with SCD exhibited a larger bone marrow volume compared with control subjects, even after standardization. CONCLUSIONS: Patients with SCD exhibited significant quantifiable changes in the craniofacial bone marrow because of failure of red-to-yellow marrow conversion and iron deposition that can be identified by qMRI relaxometry and volumetry. Both qMRI relaxometry and volumetry may be used as noninvasive tools for assessment of disease severity.

Clinical and radiologic findings of inner ear involvement in sickle cell disease.

BACKGROUND AND PURPOSE: SCD has been reported to involve the inner ear and result in LH and LO. Our purpose was to examine the prevalence of inner ear involvement and to assess the relationship between clinical and imaging findings in patients with SCD. MATERIALS AND METHODS: Review of our institution's imaging data base for patients with SCD who underwent imaging of the brain or head and neck region or both by CT, MR, or both from 2004 to 2008 was performed. Presenting symptoms, type of SCD, sex, imaging studies performed, and imaging findings were documented. RESULTS: Among 89 patients with SCD identified (41 males, 48 females), 17 patients (14 males, 3 females; 10-48 years old) underwent imaging evaluation for inner ear complaints, including SNHL, dizziness, vertigo, and tinnitus. LH was identified in 3 patients (3 males) and LO was identified in another 3 patients (2 males, 1 female). All patients with LH had sickle-hemoglobin C disease, whereas those with LO consisted of 2 patients homozygous for HbS (2) and 1 with HbS/ß-thalassemia. Patients with LH presented with vestibular symptoms (2 vertigo, 1 dizziness), whereas patients with LO presented with SNHL. LH was seen in the basal turn of cochlea and vestibule, whereas LO involved the lateral semicircular canal. CONCLUSIONS: LH and LO were found in approximately one-third of patients with SCD with inner ear symptoms and preferentially affected males.

Fetal hemoglobin in sickle cell anemia: genetic determinants of response to hydroxyurea.

The increase in fetal hemoglobin (HbF) in response to hydroxyurea (HU) varies among patients with sickle cell anemia. Twenty-nine candidate genes within loci previously reported to be linked to HbF level (6q22.3-q23.2, 8q11-q12 and Xp22.2-p22.3), involved in metabolism of HU and related to erythroid progenitor proliferation were studied in 137 sickle cell anemia patients treated with HU. Three-hundred and twenty tagging single nucleotide polymorphisms (SNPs) for genotyping were selected based on HapMap data. Multiple linear regression and the nonlinear regression Random Forest method were used to investigate the association between SNPs and the change in HbF level after 2 years of treatment with HU. Both methods revealed that SNPs in genes within the 6q22.3-23.2 and 8q11-q12 linkage peaks, and also the ARG2, FLT1, HAO2 and NOS1 genes were associated with the HbF response to HU. Polymorphisms in genes regulating HbF expression, HU metabolism and erythroid progenitor proliferation might modulate the patient response to HU.

Dominantly inherited beta thalassaemia intermedia caused by a new single nucleotide deletion in exon 2 of the beta globin gene: Hb morgantown (beta91 CTG>CG).

Family members in multiple generations of an Irish-American family were investigated for moderate to severe microcytic anaemia, inherited in an autosomal dominant fashion. A novel frameshift mutation of the beta globin gene was discovered. This study highlights the importance of considering dominantly inherited beta thalassemia in the investigation of anaemia, even in patients with ethnic backgrounds not usually associated with beta thalassaemia.

Predicting clinical severity in sickle cell anaemia.

The ability to predict the phenotype of an individual with sickle cell anaemia would allow a reliable prognosis and could guide therapeutic decision making. Some risk factors for individual disease complications are known but are insufficiently precise to use for prognostic purposes; predicting the global disease severity is not yet possible. Genetic association studies, which attempt to link gene polymorphisms with selected disease subphenotypes, may eventually provide useful methods of foretelling the likelihood of certain complications and allow better individualized treatment.

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.

Pathophysiology of sickle cell disease: role of cellular and genetic modifiers.

Sickle hemoglobin (HbS), caused by a point mutation in the beta-globin gene of hemoglobin, polymerizes when deoxygenated. The pathophysiology of sickle cell disease results from cellular defects caused directly by the hemoglobin mutation interacting with the environment and many other gene products--a few known, but most yet unidentified--a typical example of epistasis. How normal tissue perfusion is interrupted is complex and why the phenotype of sickle cell disease differs from patient to patient is poorly understood. We review the "classic" aspects of the pathophysiology of sickle cell disease and focus on known and potential modulators of the phenotype of this disorder.

Modulation of fetal hemoglobin in sickle cell anemia.

A partial understanding of the pathophysiology of sickle cell disease has suggested one means of treatment-increasing the distribution and concentration of fetal hemoglobin in sickle erythrocytes. Although this can be accomplished clinically with drugs like hydroxyurea, a complete understanding of the molecular and cellular basis of fetal hemoglobin regulation may suggest new and better ways of attaining this goal.

Gene expression profiling during erythroid differentiation of K562 cells.

We studied the temporal changes in gene expression in K562 cells at intervals from 2 to 48 h following induction using differential display polymerase chain reaction and gene expression arrays. More than 110 cDNA fragments representing 86 unique mRNAs were either up- or downregulated during erythroid differentiation. Sixty-one of the differentially expressed cDNA fragments had more than 95% homology to known GenBank sequences; 21 represented cDNA sequences with only dbEST or high-throughput gene-screening database matches. Four fragments had no database matches. Using gene expression arrays, 73 differentially expressed genes were observed. Unique expressed sequence tags (ESTs) were used to "clone" two novel genes from available databases and their tissue expression was examined. Erythroid maturation in induced K562 cells is associated with differential expression of many genes. Some differentially expressed clones were transcription factors and 25 expressed fragments with open reading frames were found whose function remains unknown.

Role of epistatic (modifier) genes in the modulation of the phenotypic diversity of sickle cell anemia.

Sickle hemoglobin is the product of one mutated gene, but the disease phenotype is the product of many genes. Polymorphism among the genes responsible for the pleotropic effects can be epistatic (or modifier) genes contributing to interindividual variation that characterizes sickle cell anemia patients. Modulation in the hemoglobin F levels is associated with the beta-globin gene cluster haplotypes and to gender and chromosomal sites different from chromosome 11 influencing the severity of the disease. Coexistence of alpha thalassemia with sickle cell disease produces hematologic and clinical consequences that are beneficial in some complications but deleterious in others. There is little if any modulation of the phenotype of sickle cell anemia by coexistence of G6PD deficiency. Mutations that favor blood coagulation or thrombosis may influence the phenotype of the disease. Improved understanding of the influence of genes involved in modulating the complex pathophysiology of sickle cell disease may allow prediction of the phenotype of sickle cell patients and aid in management decisions.

Maximum urine concentrating ability in children with Hb SC disease: effects of hydroxyurea.

Studies in adults with Hb SC disease suggested that hydroxyurea reduced hemolysis and increased red cell hydration. Because increased hydration should diminish the polymerization tendency of Hb S we hypothesized that hydroxyurea might repair the urine concentration defect of HbSC disease. Eight Hb SC disease patients, aged 10 to 17 years, were given hydroxyurea daily. Maximal urine concentrating ability following overnight fasting and after subcutaneous arginine vasopressin (dDAVP), blood counts, and cell volumes were observed for 12-15 months. All patients had impaired urine concentrating ability prior to hydroxyurea treatment and failed to increase their ability to concentrate urine following treatment (maximum urine concentration after an overnight fast and dDAVP, 520-530 mOsm). Mean corpuscular volume (MCV) and reticulocyte MCV increased after administration of hydroxyurea, and the reticulocyte count and ratio of red cell hemoglobin to reticulocyte hemoglobin fell but there was little change in PCV. Hb F increased substantially in 2 patients but showed little change in the remaining patients. There was no evidence that hydroxyurea was associated with increased urine concentrating ability in children with Hb SC disease. These results may reflect irreversible renal medullary damage prior to beginning treatment or insufficient intensity or duration of treatment.

Induction of globin synthesis in K562 cells is associated with differential expression of transcription factor genes.

Globin gene switching may be mediated by proteins expressed during different stages of development. Their identification may clarify the mechanisms of the conversion from fetal to adult globin production and lead to new approaches to reversing or retarding the gamma- to beta-globin gene switch. To explore this hypothesis, K562 erythroleukemia cells were induced to differentiate with 1.25, 2.5, and 5 mM sodium butyrate and gene expression was studied after 24, 48, and 72 h. Erythroid differentiation was verified by benzidine staining and by measuring the activity of a transduced A gamma-globin gene promoter linked to a luciferase reporter gene. Using differential display polymerase chain reaction (PCR), total mRNA extracted from induced cells at each time point of induction was reverse transcribed in the presence of A, G, and C anchored primers and 16 arbitrary primers, calculated to amplify approximately 50% of expressed genes. Amplified mRNAs from induced and uninduced cells were separated in polyacrylamide gels and compared. More than 110 cDNA fragments which appeared to represent either up- or downregulated mRNA species in induced K562 cells were identified. Sixty-four of these fragments had more than 95% homology to known GenBank sequences. Seventeen fragments with characteristics of transcription factors were cloned. These include differentiation-related gene-1 (drg-1), PAX 3/forkhead transcription factor, HZF2 which is a Kruppel-related zinc finger protein, three helix-loop-helix proteins (heir-1, Id3, and GOS8), alpha-NAC transcriptional coactivator, LIM domain protein, and trophoblast hypoxia regulating factor. Differential expression of all 17 fragments over 72 h was confirmed by reverse Northern dot blot analysis, semiquantitative PCR using nested primers, and Northern analysis. Erythroid maturation in induced K562 cells is associated with differential expression of numerous genes. Some encode transcription factors that could effect the initiation of HbF synthesis. Almost half of the differentially expressed clones contained cDNAs of unidentified open reading frames and these are the object of continued study.

Low-density lipoprotein susceptibility to oxidation and cytotoxicity to endothelium in sickle cell anemia.

Patients with sickle-cell anemia exhibit pro-oxidative metabolic perturbations. We hypothesize that because of chronic oxidative stress, plasma low-density lipoprotein (LDL) from patients with sickle-cell anemia is more susceptible to oxidation. To test this hypothesis, LDL susceptibility to copper-mediated oxidation was measured in 24 patients with sickle-cell anemia and 48 control subjects. Sickle-cell LDL was more susceptible to oxidation than control LDL, measured by a 22% shorter mean lag time between LDL exposure to CuSO4 and conjugated diene formation (97 vs 124 minutes; P = .023). LDL vitamin E, iron, heme, and cholesterol ester hydroperoxide (CEOOH) levels were also measured. LDL vitamin E levels were significantly lower in patients with sickle-cell anemia compared with control subjects (1.8 vs 2.9 mol/mol LDL; P = .025), but there was no correlation with lag time. Pro-oxidant heme and iron levels were the same in sickle-cell and control LDL. LDL CEOOHs were not significantly different in sickle and control LDL (3.1 vs 1.2 mmol/mol of LDL unesterified cholesterol, P = .15), but LDL CEOOH levels were inversely correlated with lag times in patients with sickle-cell anemia (r2 = 0.38; P = .018). The cytotoxicity of partially oxidized LDL to porcine aortic endothelial cells was inversely correlated with lag times (r2 = 0.48; P = .001). These preliminary data suggest that increased LDL susceptibility to oxidation could be a marker of oxidant stress and vasculopathy in patients with sickle-cell anemia.

Sickle cell anemia as a possible state of enhanced anti-apoptotic tone: survival effect of vascular endothelial growth factor on circulating and unanchored endothelial cells.

The biologic processes of apoptosis and angiogenesis are linked in endothelial biology because some endothelial cell growth factors also exert anti-apoptotic effects. We studied whether apoptosis is occurring in circulating endothelial cells (CEC) that have lost the survival signals derived from anchorage to extracellular matrix. Consistent with this expectation, 64% +/- 16% of CEC from normal donors showed evidence of apoptosis (by morphology and TdT-mediated dUTP nick end labeling [TUNEL] assay). However, only 30% +/- 15% (P <.001 v normal) of CEC from donors with sickle cell anemia were apoptotic. Vascular endothelial growth factor (VEGF) levels were significantly (P =.001) higher in plasma of sickle donors (120.1 +/- 81.4 pg/mL) than that of normal donors (37.6 +/- 34.6 pg/mL), and there was an inverse correlation between VEGF and CEC apoptosis (r =. 612, P =.001). Consistent with stimulation by VEGF, CEC from sickle donors exhibited increased expression of alphavbeta3. In vitro experiments showed that VEGF inhibits apoptosis for cultured endothelial cells that are kept unanchored and not allowed to re-establish attachment to extracellular matrix, thus demonstrating that VEGF provides survival signals independent of its ability to promote matrix reattachment. These data suggest the hypothesis that sickle cell anemia is a state of enhanced anti-apoptotic tone for endothelial cells. If true, this has implications for disease pathobiology, particularly the development of neovascularizing retinopathy.