Betsy Clair

Genetic risk factors for cerebrovascular disease in children with sickle cell disease: design of a case-control association study and genomewide screen

BackgroundThe phenotypic heterogeneity of sickle cell disease is likely the result of multiple genetic factors and their interaction with the sickle mutation. High transcranial doppler (TCD) velocities define a subgroup of children with sickle cell disease who are at increased risk for developing ischemic stroke. The genetic factors leading to the development of a high TCD velocity (i.e. cerebrovascular disease) and ultimately to stroke are not well characterized.MethodsWe have designed a case-control association study to elucidate the role of genetic polymorphisms as risk factors for cerebrovascular disease as measured by a high TCD velocity in children with sickle cell disease. The study will consist of two parts: a candidate gene study and a genomewide screen and will be performed in 230 cases and 400 controls. Cases will include 130 patients (TCD ≥ 200 cm/s) randomized in the Stroke Prevention Trial in Sickle Cell Anemia (STOP) study as well as 100 other patients found to have high TCD in STOP II screening. Four hundred sickle cell disease patients with a normal TCD velocity (TCD < 170 cm/s) will be controls. The candidate gene study will involve the analysis of 28 genetic polymorphisms in 20 candidate genes. The polymorphisms include mutations in coagulation factor genes (Factor V, Prothrombin, Fibrinogen, Factor VII, Factor XIII, PAI-1), platelet activation/function (GpIIb/IIIa, GpIb IX-V, GpIa/IIa), vascular reactivity (ACE), endothelial cell function (MTHFR, thrombomodulin, VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1), inflammation (TNFα), lipid metabolism (Apo A1, Apo E), and cell adhesion (VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1). We will perform a genomewide screen of validated single nucleotide polymorphisms (SNPs) in pooled DNA samples from 230 cases and 400 controls to study the possible association of additional polymorphisms with the high-risk phenotype. High-throughput SNP genotyping will be performed through MALDI-TOF technology using Sequenoms MassARRAY™ system.DiscussionIt is expected that this study will yield important information on genetic risk factors for the cerebrovascular disease phenotype in sickle cell disease by clarifying the role of candidate genes in the development of high TCD. The genomewide screen for a large number of SNPs may uncover the association of novel polymorphisms with cerebrovascular disease and stroke in sickle cell disease.

The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, βS‐globin gene haplotype, co‐inherited α‐thalassemia trait and Hb F on steady‐state serum bilirubin levels in sickle cell anemia

Abstract:  Purpose: Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers –βS‐globin gene haplotype, Hb F, co‐inherited α‐thal trait, age and gender. Methods: The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. Results: There were 67 SCA patients (41 males and 26 females), aged 2–44 yr (mean of 20.6 ± 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 ± 1.5, 3.8 ± 2.3 and 5.6 ± 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = −0.304, P = 0.016) of serum bilirubin with Hb F. The β‐globin haplotype and co‐existing α‐thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. Conclusions: Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.

Acute splenic complications and implications of splenectomy in hemoglobin SC disease.

Splenectomy indications and outcome were evaluated in 124 adults with hemoglobin SC disease (Hb SC). Twelve patients (9.6%) required splenectomy. There was a significant difference between the splenectomy group and the non‐splenectomy group, respectively, regarding Hb levels (median 7.2 g/dL vs. 12.5 g/dL, P < 0.0001), platelet counts (median 146 × 106/L vs. 275 × 106/L, P = 0.031), palpable spleen rate (66% vs. 16%, P = 0.0003%), acute chest syndrome frequency (75% vs. 12%P = 0.0004) and cholecystectomy rate (66% vs. 13%, P = 0.0004). No significant morbidity or mortality occurred postsplenectomy. There is a subgroup of Hb SC patients requiring splenectomy, in which splenectomy is effective. Although it appears to be safe regarding short‐term complications of surgery, long‐term adverse effects such as infections have to be evaluated cautiously.

A Novel Approach to Rapid Determination of βS‐Globin Haplotypes: Sequencing of the Aγ‐IVS‐II Region

β‐Globin gene cluster haplotypes were originally determined by restriction endonuclease mapping with Southern blots of polymorphic sites around the gene cluster. Over the years, haplotyping has been found to be useful, not only in population genetics but also in predicting the severity of hemoglobinopathies such as sickle cell disease. The sickle mutation occurs on five distinct haplotypes. The hitherto used methods are cumbersome and time‐consuming, making haplotype determination a tedious procedure. We report our experience with a novel, rapid approach to haplotyping based on sequence polymorphisms in the Aγ‐IVS‐II region. We provide an algorithm that allows rapid assignment of the four African haplotypes carrying the sickle mutation.