Research projects by investigators associated with the Boston Comprehensive Sickle Cell Center and the Center of Excellence in Sickle Cell Disease are focused on disorders of red blood cells with a special emphasis on inherited disorders of hemoglobin.

Fetal hemoglobin (HbF): Severe β thalassemia is a devastating disease of public health magnitude in many parts of the world. HbF can modulate the severity of this disorder by compensating for the shortfall of normal β-globin chain production, thereby improving the globin chain imbalance and total hemoglobin level. The capability to produce HbF varies, and this variation is a multigenic trait explained by genetic lab-imageheterogeneity in cis-acting elements and trans-acting factors modulating γ-globin gene expression; genes or loci controlling erythroid cell differentiation and proliferation; genes or loci that directly or indirectly modulate HbF concentration. To define genetic modifiers of HbF expression in β thalassemia in the southern Chinese population and in Thailand, we are identifying informative SNPs and haplotype structures in candidate genes, using family triads where parents are thalassemia carriers, discovering SNPs, haplotypes and genes that are associated with HbF/F-cells levels in thalassemia heterozygotes and correlating the results of these analyses to disease severity in patients who are either homozygous or compound heterozygous for β thalassemia. We evaluated demographic, clinical, laboratory, and genetic characteristics in 241 unrelated adult thalassemia carriers in Hong Kong. They had wide variations in Hb F and F-cell numbers skewing toward higher levels. Individuals who co-inherited the Xmn T-allele in the HBG promoter had higher HbF and more F-cells compared with those lacking this allele. The heritabilities of HbF and F-cells were calculated in 66 families to be 0.7 to 0.9.

Genome-wide association studies (GWAS) are also ongoing. SNPs in BCL11A on chromosome 2p were associated with HbF level in Chinese and Thais with either β thalassemia or HbE trait, and in African Americans with sickle cell anemia. These results confirmed the association reported in normal Europeans, Sardinians with β thalassemia and another group of African Americans with sickle cell anemia. They also suggested that the functional motifs responsible for modulating HbF reside within a 3 kb region in the second intron of BCL11A.

In other studies focused on HbF, a novel polymorphism of the γ-globin gene, altering a presumptive GATA-1 binding site was hypothesized to act as a negative regulatory element, possibly through interaction with a nuclear protein complex containing GATA-1. DNA-protein centrifugebinding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt -675 and -526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by 2- to 3-fold. The binding motif is uniquely conserved in simian primates with a fetal pattern of γ-globin gene expression. These results suggest that the GATA motif has a functional role in silencing γ–globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of gamma-globin gene expression in adults.

HbF inhibits the polymerization of HbS and is, therefore, an important modulator of the phenotype of sickle cell anemia. In this disease, HbF concentrations span two orders of magnitude. Hydroxyurea can prevent some of the vasoocclusive complications of sickle cell anemia, an effect that is due, at least partially, to its ability to increase HbF levels. But, patients who take hydroxyurea to increase HbF levels have an unpredictable response.

genetic modulation of sickle cell disease

To define this genetic heterogeneity, we are discovering SNPs and haplotypes that predict the HbF level in sickle cell anemia patients taking hydroxyurea and SNPs and haplotypes associated with baseline HbF concentrations. We have examined SNPs in candidate genetic loci that plausibly modulate the HbF response to hydroxyurea and baseline HbF concentration, additional biologically important genes. We hope ultimately to discover SNPs, haplotypes or genes that predict or modulate the HbF response to hydroxyurea and better define HbF regulation in sickle cell anemia. These studies are now being done by GWAS in 2 patient groups, examining both baseline HbF and the HbF response to hydroxyurea.

Genetic modulation of sickle cell disease: Homozygosity for a single β-globin gene mutation (HBB; glu6val) causes sickle cell anemia. Nevertheless, its exceptional phenotypic variability of HbSS suggests that other genes modulate its phenotype. Some disease-modulating genes have been discovered.

We are involved in an integrated approach to further to probe the genetic modulation of sickle cell anemia by: 1. a continued quest for novel modifying genes using GWAS and extending our work in sickle cell disease to other vascular disorders and exceptional logevity to understand the commonalities among these conditions; 2. examining gene expression in sickle leukocytes and blood outgrowth endothelial cells; and 3. searching for modified plasma proteins associated with the phenotype of sickle cell pulmonary hypertension. Advanced genotyping, gene expression and proteomics methods, coupled with novel analytical tools are used to accomplish our goals.

genetic modulation of sickle cell anemia

Intravascular hemolysis in sickle cell anemia could contribute to complications associated with nitric oxide deficiency, advancing age, and with increased mortality. We have used the distribution of serum lactic dehydrogenase (LDH) values as a surrogate measure of intravascular hemolysis and defined chronic hyper-hemolysis as the top LDH quartile, and compared this to the lowest LDH quartile.

Hyper-hemolysis subjects had higher systolic blood pressure, higher prevalence of leg ulcers, priapism and pulmonary hypertension, while osteonecrosis and pain were less prevalent. Hyper-hemolysis was influenced by fetal hemoglobin and α thalassemia, and was a risk factor for early death. These results suggest that an important class of disease modifiers in sickle cell anemia affect the rate of hemolysis. Individuals with sickle cell disease have been shown to have endothelial dysfunction. While this might result from decreased bioavailable nitric oxide (NO) due to hemolysis, endothelial dysfunction could result from an aldosterone-mediated decrease in glucose-6-phosphate dehydrogenase (G6PD) activity.Glucose-6-Phosphate Dehydrogenase Modulation of Endothelial Function in Sickle Cell Disease:

hemolysis analysis

G6PD, the first and rate-limiting enzyme of the pentose phosphate pathway, is the principal intracellular source of NADPH, which serves as a reducing equivalent and a cofactor for the endothelial isoform of nitric oxide synthase. G6PD regulates endothelial function by modulating cellular redox state and NO production. In sickle cell disease, systemic dehydration due to renal dysfunction is common; (sub)acute or chronic dehydration activates the renin-aldosterone system and clinical studies have shown that patients have elevated levels of renin and aldosterone. Hyperaldosteronism has been associated with endothelial dysfunction and aldosterone decreases G6PD expression and activity in endothelial cells in vivo, resulting in increased oxidant stress, decreased bioavailable NO, and impaired vascular reactivity.This project examines the molecular and cellular mechanisms by which an aldosterone-mediated decrease in G6PD activity influences endothelial cell function under steady-state, acute hypoxia, and dehydration conditions in cells isolated from mouse models of sickle cell disease and G6PD deficiency; determines the functional consequences of decreased G6PD activity in sickle cell disease by evaluating indices of oxidant stress and bioavailable NO in the presence or absence of cell-free hemoglobin; examines the efficacy of an aldosterone antagonist, or G6PD over-expression in improving endothelial function in vitro; in mouse models of sickle cell disease and G6PD deficiency, under steady-state, acute hypoxia, and dehydration conditions, examines endothelial function, NO bioavailability, oxidant stress and vascular reactivity, and examines polymorphisms that may be associated with this important complication.

Modeling of interactions among genes and phenotypes: The genetic dissection of a complex trait requires the ability to disentangle the web of interactions among genes, environment and phenotype. To model these relationships, we are developing multivariate analysis using Bayesian networks, multivariate dependency models that account for simultaneous associations and interactions among multiple genes and their interplay with clinical and physiological factors.

Bayesian Network of SNPs Associated with CVA

A Bayesian network is a directed acyclic graph in which nodes represent random variables and arcs define directed stochastic dependencies quantified by probability distributions. Their modular nature makes these networks ideal tools for the analysis of large association studies like the work on genotype-phenotype associations in sickle cell disease.

Furthermore, these networks can be used for prognosis: a network capturing the relationship between genotypes and phenotype can be used to compute the probability that a new individual with a particular genotype will manifest the phenotype. Other analytical methods like classification and regression trees are also being applied to the genetic studies described. Clinical studies are: 1. examining the ability of pulse butyrate to increase HbF and F-cells in patients with sickle cell anemia; 2. using hydroxyurea plus magnesium in HbSC disease to study the effects on cell density and pain; 3. evaluating P-selectin blockade on microvascular blood flow; 4. testing if Losartin can prevent renal functional impairment in sickle cell disease; and 5. seeing if serum phospholipase A2 levels in patients admitted with acute painful episodes can be used as a guide to prophylactic transfusion to prevent acute chest syndrome.

Clinical trials of new disease treatments and patients’ adherence to treatment: Clinical studies are: 1. examining the ability of pulse butyrate to increase HbF and F-cells in patients with sickle cell anemia; 2. using hydroxyurea plus magnesium in HbSC disease to study the effects on cell density and pain; 3. evaluating P-selectin blockade on microvascular blood flow; 4. testing if Losartin can prevent renal functional impairment in sickle cell disease; and 5. seeing if serum phospholipase A2 levels in patients admitted with acute painful episodes can be used as a guide to prophylactic transfusion to prevent acute chest syndrome.

Structure, function and synthesis of abnormal hemoglobins: Our Hemoglobin Diagnostic Laboratory  is constantly discovering interesting hemoglobin variants that increase our understanding of the biological and clinical effects of globin gene mutations.